Vessel for cultivating cells in a liquid medium in a cultivation procedure

Abstract

The present invention relates to the field of biotechnology, specifically to cultivating cells which can be used for cell therapy. The present invention provides a vessel for cultivating cells in a liquid medium in a cultivation procedure, wherein the vessel comprises a casing defining a main volume, and a reservoir defining a reservoir volume, wherein the reservoir volume and the main volume are adjacently arranged and are configured to be fluidically connected for fluidic exchange, characterized in that the reservoir is a pre-shaped reservoir. The present invention further provides a system for cultivating cells in a liquid medium in a cultivation procedure. The present invention further provides a method for cultivating cells in a liquid medium and / a method for manufacturing a cell therapy product. The present invention further provides use of the vessel and / or system for manufacturing a cell therapy product

Description

[0001] VESSEL FOR CULTIVATING CELLS IN A LIQUID MEDIUM IN A

[0002] CULTIVATION PROCEDURE

[0003] TECHNICAL FIELD OF THE INVENTION

[0004] The present invention relates to the field of biotechnology, specifically to cultivating cells which can be used for cell therapy. The present invention provides a vessel for cultivating cells in a liquid medium in a cultivation procedure, wherein the vessel comprises a casing defining a main volume, and a reservoir defining a reservoir volume, wherein the reservoir volume and the main volume are adjacently arranged and are configured to be fluidically connected for fluidic exchange, characterized in that the reservoir is a pre-shaped reservoir. The present invention further provides a system for cultivating cells in a liquid medium in a cultivation procedure. The present invention further provides a method for cultivating cells in a liquid medium and / a method for manufacturing a cell therapy product. The present invention further provides use of the vessel and / or system for manufacturing a cell therapy product

[0005] BACKGROUND OF THE INVENTION

[0006] In the field of biotechnology, cultivating cells is essential for several applications. Here, the cells are usually cultivated in different kinds of bioreactors, wherein a common type of bioreactors is so-called rocking motion bioreactors. In a rocking motion bioreactor, a cultivation procedure is performed by using a vessel, which is moved for a period of time in a rocking motion using a rocker platform of the bioreactor. Due to this controlled movement of the vessel, cells within a liquid medium in the vessel can be cultivated under enhanced conditions, in particular by providing well-mixed conditions with an adequate oxygen supply. Rocking motion bioreactors may be used in the area of cell and gene therapy. Here, the expansion of genetically modified T-cells for example may be performed within a vessel that is used with a rocking motion bioreactor.

[0007] Manufacturing of a cell therapy product conventionally involves expanding the cells in an expansion process to generate a sufficiently high cell number. Hence, during this process, the cells may have to undergo several cultivation and transfer steps involving culture vessels of increasing size and thus volume. Using several vessels, for example a preliminary vessel with a smaller volume and a subsequent vessel with a larger volume, is disadvantageous for several reasons, including that changes in cell environment on transition between cultivation chambers cause negative effects on cells, with several vessels additional labware is required for cultivation, additional space is required, the cells may be damaged by shear during the transfer, the transfer is error-prone and has a risk for contamination.

[0008] WO 2018 / 077994 Al relates to a tray configured to be provided on a base station of a bioreactor system. As a cell culture vessel, WO 2018 / 077994 Al provides a casing in form of a bag, wherein the casing defines a main volume for receiving liquid medium containing the cells in a subsequent cultivation phase. When the casing on the tray is mounted to a rocker platform, the flexible vessel develops a reservoir due to an inner bottom part of the tray that is movable into a lower position. The formed reservoir defines a reservoir volume for receiving liquid medium containing cells in the preliminary cultivation phase, while movement of the bottom part of the tray into a planar position removes presence of the reservoir, such that only the main volume remains. When using such a bioreactor system, during the handling of a small volume of liquid medium in the preliminary cultivation phase, the liquid medium containing the cells is located mostly in the reservoir volume. Here, evaporation is minimized due to a small evaporation surface. Also, the nutrient supply is enhanced because of better mixing of the liquid medium containing the cells. During the handling of a larger volume of liquid medium in the subsequent cultivation phase, the liquid medium containing the cells in the main volume, as the reservoir volume diminishes due to movement of the bottom part into a planar position.

[0009] Even if using the known vessel already enhances the cultivation procedure and thus cultivating of cells in the liquid medium, there is still the need for further improvement. For example, the reservoir of the vessel is only developed when properly mounted to the rocker platform. The rocker platform thus comprises a plane portion and a recessed portion, in which the casing of the vessel is partly inserted such that the reservoir is developed. Here, a high degree of accuracy is needed during the mounting of the vessel on the rocker platform and the achievable consistency of the reservoir shape and thus volume is challenging to achieve. Furthermore, the mechanical elevation mechanism of the bottom part of the tray adds complexity to the overall system. Besides that, development of the reservoir itself may lead to stress on the vessel, since the material of the vessel might be deformed to a certain degree to fit into the respective portion of the rocker platform. This may lead to ceases and / or damages of the vessel, like ruptures, leaks or the like. Furthermore, the shape of the reservoir and thus the reservoir volume is mainly determined and limited by the recess portion, the transition of the recess portion and the plane portion as well as the properties of the vessel itself like its flexibility. Further, measurements of certain properties of the cells and / or the liquid medium might still be challenging by using the known vessel.

[0010] It is an object to provide a vessel which can be handled with less complexity and is still applicable or even optimized for different volumes.

[0011] SUMMARY OF THE INVENTION

[0012] The invention is based on the problem of providing a vessel for cultivating cells in a liquid medium in a cultivation procedure, which is optimized for several volumes and easy to mount, use or the like. The invention is further based on the problem of providing a vessel for cultivating cells in a liquid medium in a cultivation procedure, which is optimized for culturing different volumes and reduced complexity.

[0013] Specifically, the invention provides a vessel for cultivating cells in a liquid medium in a cultivation procedure comprising a casing defining a main volume, and a reservoir defining a reservoir volume, both of which are adjacently arranged and are configured to be fluidically connected for fluidic exchange. Furthermore, the reservoir is characterized in that it is a pre-shaped reservoir. Such vessel including the pre-shaped reservoir makes it easy to handle and dimensionally stable. This achieves a high reproducibility for a critical process, such as cell cultivation, particularly for cell or gene therapy manufacturing. Because of the pre-shaped reservoir, which may correlate with a pre-shaped reservoir volume, several advantages regarding evaporation, nutrient supply and / or measurement occur. It also becomes easier to drain the vessel fully, for example via ports attached to the reservoir. Furthermore, the configuration allows for increasing the cell culture volume throughout culture. This is particularly advantageous throughout the expansion process of cells, wherein the cell number is increased. For example, during the cultivation procedure the volume of the liquid medium in the vessel increases over time. For instance, in a preliminary cultivation phase the volume of the liquid medium is rather small compared to the volume in a subsequent cultivation phase. Here, the liquid medium containing the cells can be held mainly, preferably completely, in the reservoir. As a result, evaporation might be minimized or at least reduced due to an optimized surface to volume ratio of the liquid medium. However, it is possible to further control evaporation and condensation, for example by using a condensate arrangement. Furthermore, the nutrient supply might be improved since mixing of the liquid medium in the reservoir is improved. If needed, also measuring properties of the cells and / or the liquid medium can be improved. Since the liquid medium can be held mainly in the reservoir in the preliminary cultivation phase, accurate measurement might be possible even with small volumes of liquid medium containing the cells. A preferred reservoir-shape also ensures liquid contact with only a small surface area of reservoir material. This ensures reduced movement of any leachables or extractables from the reservoir material, because this movement is driven by surface area in contact. Nevertheless, in the case the cell number has to be increased throughout culture, the vessel volume can be increased exceeding the reservoir and utilizing the main volume of the casing. Hence, an advantageous environment for the scale-up is provided without the requirement to change the vessel. Such advantages of the vessel are highlighted in the Examples (see e.g., Figs. 7 and 8).

[0014] Hence, according to a first aspect of the invention, a vessel for cultivating cells in a liquid medium in a cultivation procedure is provided, wherein the vessel comprises a casing defining a main volume, and a reservoir defining a reservoir volume, wherein the reservoir volume and the main volume are adjacently arranged and are configured to be fluidically connected for fluidic exchange, characterized in that the reservoir is a pre-shaped reservoir.

[0015] According to a second aspect of the invention, a system for cultivating cells in a liquid medium in a cultivation procedure is provided, wherein the system comprises a vessel according to the first aspect of the invention.

[0016] According to a third aspect of the invention, a method for cultivating cells in a liquid medium is provided comprising: a) providing cells in a liquid medium; b) cultivating the cells using a vessel according to the first aspect of the invention and / or a system according to the second aspect of the invention.

[0017] According to a fourth aspect of the invention, a method for manufacturing a cell therapy product is provided comprising: a) providing cells in a liquid medium; b) cultivating the cells using a vessel according to the first aspect of the invention and / or a system according to the second aspect of the invention. According to a fifth aspect of the invention, use of the vessel according to the first aspect of the invention and / or system according to the second aspect of the invention for manufacturing a cell therapy product is provided. Preferably, wherein manufacturing comprises at least one of the following: activating cells, preferably activating T cells; transducing cells, preferably transducing T cells; transfecting cells, preferably transfecting T cells; and / or expanding cells, preferably T cells, more preferably genetically modified T cells.

[0018] According to a sixth aspect of the invention, use of the vessel according to the first aspect of the invention and / or system according to the second aspect of the invention in one of the methods disclosed herein is provided.

[0019] According to a further aspect of the invention, cultivated cells are provided obtained by the method according to the third aspect of the invention.

[0020] According to a further aspect of the invention, a cell therapy product is provided obtained by the method according to the fourth aspect of the invention.

[0021] BRIEF DESCRIPTION OF THE FIGURES

[0022] In the following, embodiments of the invention are explained with respect to the drawings. The drawings show in

[0023] Fig. 1 a system as proposed comprising a rocker platform in a perspective view and the rocker platform with a vessel as proposed in a partly exploded view,

[0024] Fig. 2 the vessel according to fig. 1 in a top perspective view (a) and a bottom perspective view (b),

[0025] Fig. 3 the vessel according to fig. 1 in a respective sectional view along the intersection line according to fig. 2a) in the directions A-A (a), B-B (b) and C-C (c),

[0026] Fig. 4 another embodiment of the proposed vessel and schematically a loop connected to the tubes of the vessel,

[0027] Fig. 5 the vessel of fig. 4 in a view partially from below and a side,

[0028] Fig. 6 a top view inside the vessel of fig. 4 with the upper casing part removed,

[0029] Fig. 7 the total cell number (see 1) and daily fold expansion (see 2) of T cells using the bioprocessing system (BRO3) compared to a reference cell culture (BRO2), Fig. 8 the viable cell density using the bioprocessing system (BRO3) compared to a reference cell culture (BRO2) during the whole culture (see 1) and the perfusion culture only (see 2),

[0030] Fig. 9 the vessel comprising a binding unit,

[0031] Fig. 10 the total cell number (see 1), total fold expansion (see 2), cell viability (see 3), percentage of activated CD4+ T cells (see 4) and percentage of activated CD8+ T cells (see 5) using T cells from two different donors (BRO1 and BRO3) using the bioprocessing system according to the invention,

[0032] Fig. 11 the percentages of T cell populations over the activation and culturing period in the method according to the invention (BRI and BR2) and a flask culture (flask),

[0033] Fig. 12 the transfection efficiency (see A) and T cell phenotype (see B) of GFP-transfected T cells transfected according to a method of the present disclosure, and

[0034] Fig. 13 the percentage of T cells expressing a chimeric antigen receptor (CAR) and GFP, respectively, after being transduced with a method according to the invention.

[0035] DETAILED DESCRIPTION

[0036] The following description serves to deepen the understanding of the present disclosure and shall be understood to complement and be read together with the description of exemplary embodiments of the present disclosure as provided in the above section of this description. It is to be understood that this invention is not limited to the particular embodiments, methodologies, protocols and reagents described herein as these may vary within the scope set by the claims. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which is defined by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0037] In the following description, certain elements of the present invention will be described. These elements may be discussed with specific embodiments; however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples, features and particular embodiments should not be construed to limit the present invention to only the explicitly described embodiments or to the explicitly described combination of features. This description should be understood to disclose and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by this description unless the context indicates otherwise.

[0038] Vessel for cultivating cells in a liquid medium in a cultivation procedure according to a first aspect of the invention

[0039] According to a first aspect of the invention, a vessel for cultivating cells in a liquid medium in a cultivation procedure is provided, wherein the vessel comprises a casing defining a main volume, and a reservoir defining a reservoir volume, wherein the reservoir volume and the main volume are adjacently arranged and are configured to be fluidically connected for fluidic exchange, characterized in that the reservoir is a pre-shaped reservoir.

[0040] The vessel according to the first aspect can advantageously be used for cultivating cells from small to large scale. Specifically, the reservoir defines a reservoir volume, in which the cells can be initially cultivated. Such reservoir volume is generally lower than the main volume, allowing for small-scale culture. At the same time the reservoir volume by being adjacently arranged to the main volume and being fluidically connected for fluidic exchange provides access to the typically larger volume of the main volume defined by the casing. Therefore, the cells can be expanded with increasing volume in an adequate environment, i.e., initially in the reservoir volume, followed by culture in the main volume (and reservoir volume). Thereby, scale-up is achieved without the necessity to change the vessel (e.g., by transfer to a larger vessel). At the same time, the reservoir volume and main volume provide an adequate culture environment for the respective needs throughout culture. For instance, initially the cells may be present in a small volume, requiring small surface areas in order to minimize evaporation effects. This can be provided by the reservoir. Later in culture, more volume is needed for obtaining a suitably high cell number, such that the main volume provides an adequate extension of the reservoir volume. Such culture mode is particularly advantageous for manufacture of a cell therapy product, such as autologous immune cell therapy (e.g. CAR T-cells), since initially the cell number obtained from a patient sample is comparably low and requires expansion for manufacturing the cell therapy product. Thus, despite a low cell number and also a low culture volume, the cells can be cultured in the very same vessel during the complete expansion process. This not only simplifies the overall process but also makes it less prone to errors and reduces contamination risks. As is demonstrated in the Examples below, also higher daily expansion rates and cell viabilities can be achieved, such that cultivation duration can be shortened and product quality is improved (see e.g., Figs. 7 and 8). By being "pre-shaped", the reservoir is dimensionally stable such that the reservoir volume is fully developed and does not require provision of a further device or system, e.g. rocker / rocking motion platform, in order to form the reservoir. "Pre-shaped" does not exclude that the reservoir can be generally deformed, however, it is to be understood that the reservoir has a certain defined shape independent of other (external) devices or system (such as a rocker platform). It is possible that the reservoir returns to its pre-shaped form when a deformation force decreases or disappears. By being pre-shaped, the reservoir can be reproducibly and stably provided enabling a more robust cultivation process. In particular, it can be achieved to provide the same volume (here reservoir volume) for each culture batch. Furthermore, by being pre-shaped, the reservoir is more resistant to deformation and material rupture compared to a reservoir or chamber that forms only when combined with a culture platform or system, such that failure or contamination risks are reduced.

[0041] The vessel is in particular suitable for cultivating cells in a liquid medium in a cultivation procedure. For cell cultivation, the cells are conventionally provided in a liquid medium, which allows for efficient expansion and manipulation of the cells. However, the present disclosure is not limited to cells being exclusively in liquid medium. For instance, the cells may be provided together with a carrier matrix or support matrix for cultivation, wherein these are dispersed in a liquid medium. In addition, the medium may comprise in part a dissolved or undissolved gaseous phase (e.g., culture gas as disclosed herein).

[0042] A "cultivation procedure" in scope of the present disclosure shall not be limited in scope. Indeed, the skilled person is well-aware of various cultivation procedures, which are adequate in order to cultivate cells in a liquid medium. Cultivation procedures may include batch cultivation, fed- batch cultivation or perfusion culture or combinations thereof. In addition, a cultivation procedure may already be achieved if the cells are provided with an adequate environment in which they may proliferate or at least not become completely apoptotic. In one embodiment, a cultivation procedure may also include adding a fluid, such as cell culture medium or reagent, to the cells present in the vessel according to the present disclosure. According to a preferred embodiment, particularly suitable for manufacture of a cell therapy product, the cultivation procedure encompasses a preliminary cultivation phase, in which the cells are preliminarily cultivated, preferably wherein cells are predominantly cultivated in the reservoir; and a subsequent cultivation phase, in which the cells are subsequently cultivated, preferably wherein cells are cultivated in the reservoir and the casing. Such culture allows for scaling up a small cell number, such as typically only obtainable from a patient sample for autologous cell therapy, in the same vessel.

[0043] A "liquid medium" according to the present disclosure in particular relates to a composition in which the cells remain to the most extent viable. For instance, the cells may be combined with a cell culture medium for providing an environment in which the cells can proliferate. Also, the cells may be combined with a buffer or storage solution, in which the cells can be stored and / or kept essentially alive. In another embodiment, the cells may be combined with an agent (also referred to as "reagent") in order to manipulate the cells, e.g. an activation agent may be provided to activate the cells and / or a genetic modification agent may be provided to genetically modify the cells. The term "liquid medium" may herein be interchangeable used with the terms "medium", "media", "liquid media". Sometimes, also an activation agent, and / or genetic modification agent, in particular transduction and / or transfection agent, may be referred to as "liquid medium", such that the term shall encompass such compositions.

[0044] The vessel according to the present disclosure comprises a casing defining a main volume, and a reservoir defining a reservoir volume, wherein the reservoir volume and the main volume are "adjacently arranged". The skilled person is well-aware about this terminology and understands it according to the common general knowledge. Following the teaching of the present disclosure it is clear that "adjacently arranged" is typically understood as two volumes (i.e., the main volume and the reservoir volume) being arranged next to each other. This does not exclude that a further volume may be in contact to the reservoir volume and / or the main volume. Rather, by being adjacently arranged, when the volume of the culture exceeds the reservoir volume it can move into the main volume, such that the culture can be continued without the need to stop the process and / or transfer the cell culture into another vessel. The arrangement of these volumes is important in order to efficiently enlarge the volume accessible for the cell culture over time, e.g., in an expansion process. While it is not excluded that between the reservoir volume and the main volume another volume is arranged, this is less preferred, as it allows less efficient enlargement of volume accessible to the cell culture. According to one embodiment, there is no volume defined by one or more tubes between the reservoir volume and the main volume. According to a preferred embodiment, the reservoir volume and the main volume are directly adjacent.

[0045] The reservoir volume and the main volume of the vessel are further configured to be fluidically connected for fluidical exchange. "Fluidic exchange" (which may be synonymously used herein with "fluidical exchange") in this context comprises that fluid in the reservoir and fluid located in the casing can be mixed such that an exchange between the fluids located in the different volumes occurs. It further may mean that liquid medium located in the reservoir and liquid medium located in the casing can be mixed such that an exchange between the liquids located in the different volumes happens. In one embodiment, the cells may undergo a preliminary cultivation phase, wherein the liquid medium might be contained solely in the reservoir. Fluidic exchange may the occur through the gas liquid interface between the reservoir volume and the main volume, considering that gas it provided and present in the main volume. In case the culture comprises a subsequent cultivation phase (after the preliminary cultivation phase), fluidic exchange between liquid medium in the reservoir and liquid medium in the casing may happen mostly in such phase. The reservoir volume and the main volume develop the overall volume of the vessel. Also, in the preliminary cultivation phase liquid may enter the main volume, in particular during rocking. The transition between the preliminary cultivation phase and the subsequent cultivation phase may be a continuous transition if for example culture medium is added continuously and not in steps.

[0046] By being "configured to be fluidically connected for fluidic exchange", the vessel also encompasses embodiments, wherein the vessel is completely deflated (e.g. put under vacuum), which may happen when providing the vessel prior to any cultivation process in order to keep the volume of such vessel as small as possible. Nevertheless, when being inflated again with a fluid (e.g. gas and / or liquid), such vessel would be fluidically connected for fluidic exchange.

[0047] Casing

[0048] A "casing" is well-known to the person skilled in the art. Typically, a casing of a vessel refers to a bag or pouch, which is preferably flexible but may also be rigid or semi-rigid. Preferably, the casing in the present disclosure is at least partially flexible to adapt throughout culture and increasing culture volumes during expansion. The vessel according to the present invention further comprises a reservoir, which may be connected or form an integral part of the casing. Generally, the casing may be pre-shaped like the reservoir. However, it is also possible that the casing is flexible and particularly obtains its form during the cultivation procedure, for example because of rising pressure or weight inside the vessel.

[0049] The casing may preferably be a single-use bag which are widely used in the field of cell culture, for instance in single-use bioreactors or for storing or conveying cell fluids, such as cell medium, culture buffer or cell culture. Such bags may comprise at least one casing part, which may also be referred to as "wall". Hence, according to a preferred embodiment, the casing comprises a casing part. Such casing part may be folded and sealed together in order to form a bag-like structure. Typically, a bag may comprise two (large) walls (here casing parts) sealed to one another. Once expanded, they have a limited volume and remain relatively thin, which justifies the fact that they are often called 2D bags (D meaning dimensions). 3D bags are also known and typically comprise two end walls and a side wall that can be folded flat or deployed unfolded, sealed to one another, with the volume able to reach up to 3,000 liters, and even more. Hence, according to a preferred embodiment, the casing comprises two or more casing parts, such as an upper casing part and a lower casing part. Such casing parts may be connected in orderto form a bag-like structure (also referred to as 2D bags). It is also within the scope of the present disclosure that the casing comprises three casing parts, or four casing parts, or five casing parts or more than five casing parts. This may allowforming 3D casings (e.g., 3D bags).

[0050] The casing part may be composed of a multilayer film comprising a contact layer which in contact with the medium that fills the casing, a barrier layer and an outer layer which is in contact with the external environment of the casing, the three layers being connected one to each other with a tie layer. In case the casing is to be filled with a biopharmaceutical product, the contact layer should be made from a material that can be in contact with this biopharmaceutical product without causing degradation of the film and of the biopharmaceutical product. Furthermore, it must be sealable on itself. For that purpose, the material is generally selected from polyolefins, such as polyethylene. The barrier layer provides a barrier to the passage of gases such as oxygen, carbon dioxide and is typically made from ethylene vinyl alcohol (EVOH). The outer layer contributes to the mechanical strength of the casing. For that purpose, it must be sufficiently flexible to withstand high mechanical stress but not be too much stretchable in order to prevent deformation of the casing when it is filled.

[0051] According to a preferred embodiment, the reservoir is attached to the casing. Such attachment advantageously allows for providing the casing and the reservoir as a single item or piece, avoiding manual assembly. Preferably the reservoir is attached to a casing part, more preferably a lower casing part. Such lower casing part may come into contact with a bioprocessing system or device, such as a rocker platform, during cultivation. Hence, such lowercasing part is preferably configured to be arranged at or on a bioprocessing system or device for cultivation, in particular a rocker platform. In the embodiment, wherein the reservoir is attached to a casing part, the casing part would have an opening allowing for the volume defined by the reservoir (i.e., the reservoir volume) to be adjacently arranged to the volume defined by the casing (i.e., main volume) and being configured for fluidic exchange. According to a preferred embodiment, the reservoir is formed integrally with the casing, preferably with a casing part, more preferably with a lower casing part. By being integrally formed, robust connection between the casing and the reservoir can be achieved. The skilled person is aware of various methods and processes for attaching or integral formation of the casing and the reservoir, and the present disclosure shall not be limited in this respect. For instance, the reservoir is attached to the casing by welding or formed integrally with the casing, in particular by injection molding and / or thermoforming and / or gluing, for example UV cured gluing.

[0052] Filter

[0053] According to a preferred embodiment, the vessel comprises at least one filter and / or is fluidically connected to at least one filter. The term "filter" is well-known by the person skilled in the art. A "filter" is typically used to remove particles from a fluid process stream and comprises a porous medium. A filtration process is a process by which particles are removed from a fluid by passing the fluid through a porous medium. Herein, the filter is particularly used in order to retain the cells within the vessel. The remaining filtered cells may be referred to as "retentate". The fluid passing the filter essentially does not comprise the cells. The passing fluid may be referred to as "permeate". The selection of a filter is within the skills of the person of ordinary skill in the art. In particular, the skilled person is well aware of various filters for retaining cells but allowing liquid to pass. Selection of an adequate pore size or molecular cut-off is within the routine of the skilled person. Hence, the present disclosure shall not be limited to a particular filter material or pore size, insofar as the material and pore size should be suitable for cell culture.

[0054] Preferably, the filter is configured to allow liquid to flow through but essentially retain cells. This does not mean that all cells are completely retained but the majority of cells shall be retained. Also, this does not exclude that cell debris or apoptotic cells (which are oftentimes smaller in size than viable cells) pass the filter.

[0055] According to a preferred embodiment, the at least one filter comprises a filter medium selected from a surface filter and a depth filter.

[0056] According to a preferred embodiment, the at least one filter is a membrane, such as a planar membrane, a hollow fiber membrane and / or floating membrane. A floating membrane may also be referred to as "pouch membrane", in particular being fluidically connected to the outside of the vessel, especially at least to the waste container (which may also be referred to as the "waste container").

[0057] The term "membrane" is known in the art. Various membrane materials can be used, including polyvinylidene difluoride (PVDF), polyethersulfone (PES), or polysulfone (PS). In one embodiment, the membrane is a PES membrane, optionally having a cutoff of 0.5 to 5 micrometer, such as 1.2 micrometer.

[0058] According to a preferred embodiment, the at least one filter comprises a main membrane, and / or a reservoir membrane, preferably both of a main membrane and a reservoir membrane. A "main membrane" herein typically refers to a membrane that is arranged at the casing. A "reservoir membrane" herein typically refers to a membrane that is arranged at the reservoir. The reservoir membrane advantageously allows to perform a filtration process at larger scale, e.g. when the cell culture is present in the casing (and typically also the reservoir). The main membrane can therefore be advantageously used to perform medium to large volume liquid filtrations, e.g. as may be required throughout a perfusion culture.

[0059] According to a preferred embodiment, the main membrane is arranged at the casing, preferably at a casing part, more preferably at the lower casing part. By being arranged at the casing, the membrane forms part of the casing and thus cell vessel, allowing for filtration through the membrane directly at the casing. Alternatively, or in addition, the main membrane is f I uid ically connected to the casing, preferably wherein fluidic connections between the casing and the main membrane are provided, in particular wherein the fluidic connections are provided by one or more tubes. Such fluid connection allows for flexibly arranging and exchanging the membrane independent of the casing and thus independent of the vessel. Nevertheless, such main membrane that is fluidically connected to the casing, preferably wherein fluidic connections between the casing and the main membrane are provided, may also encompass integral connection such as sealing or molding between the casing and the membrane. Different configurations are in general possible within the present disclosure as is recognized by the person skilled in the art. According to a preferred embodiment, the main membrane is configured for retaining cells in a perfusion culture.

[0060] When providing a main membrane that is arranged at the casing, preferably, the casing further comprises an outlet section, which is arranged outside the main volume. Specifically, such outlet section is preferably provided at the permeate side adjacent to the main membrane. Such outlet section is advantageously able to collect the permeate and guide the permeate to an outlet port. Hence, according to one embodiment, the casing comprises an outlet section, preferably, comprising an outlet port, wherein the outlet section is preferably arranged adjacent to the main membrane. An outlet port may also be referred to as "waste port" or "permeate port". According to one embodiment, the vessel comprises a waste port for draining liquid medium, which was filtered by the main membrane.

[0061] According to a preferred embodiment, the main membrane surrounds the reservoir, is adjacent to the reservoir and / or floats within the main volume, preferably wherein the main membrane is adjacent to the reservoir. Being adjacent to the reservoir does not mean that a part of the casing is between the reservoir and the main membrane. Typically, the main membrane is arranged at the same casing side or casing part, which may throughout culture be the lower casing part. This is due to the fact, that during culture the culture liquid would typically be located on the lower casing part (within the casing) due to gravity. Hence, a filtration process, such as a perfusion process, would be most efficient, when the main membrane is located where most of the culture liquid is located which is typically close to the reservoir, i.e. adjacent to the reservoir.

[0062] According to a preferred embodiment, the main membrane is configured for removing and / or adding liquid through the main membrane but essentially retaining the cells within the main volume or preferably, within the main volume and the reservoir volume. Such configuration is particularly advantageous when performing a filtration process such as perfusion throughout culture, as it allows cells to be retained within the vessel but remove culture liquid. By adding liquid through the membrane, the membrane may be backflushed, e.g. in alternating flow. Additionally, or alternatively, liquid may be added through a supply port, arranged on the retentate side, e.g., in the casing or reservoir.

[0063] According to a preferred embodiment, the main membrane is configured for cultivating the cells in the liquid medium via perfusion in the main volume or preferably, in the main volume and the reservoir volume.

[0064] According to one embodiment, the casing comprises a main membrane, preferably, wherein the reservoir does not comprise the main membrane.

[0065] According to a preferred embodiment, the reservoir membrane is arranged at the reservoir, preferably the reservoir comprises the reservoir membrane, and / or is fluidically connected to the reservoir, preferably wherein fluidic connections between the reservoir and the membrane are provided, more preferably wherein the fluidic connections are provided by one or more tubes. The reservoir membrane advantageously allows to perform a filtration process even at small scale, e.g. when the cell culture is only present in the reservoir. The reservoir membrane can therefore be advantageously used to perform low volume liquid filtrations. By being arranged at the reservoir, the reservoir membrane forms part of the reservoir and thus cell vessel, allowing for filtration through the reservoir membrane directly at the reservoir. Alternatively, or in addition, the reservoir membrane is fluidically connected to the reservoir, preferably wherein fluidic connections between the reservoir and the reservoir membrane are provided, in particular wherein the fluidic connections are provided by one or more tubes. Such fluid connection allows for flexibly arranging and exchanging the reservoir membrane independent of the reservoir and thus independent of the vessel. Nevertheless, such reservoir membrane that is fluidically connected to the reservoir, preferably wherein fluidic connections between the casing and the main membrane are provided, may also encompass integral connection such as sealing or molding between the reservoir and the membrane. Different configurations are in general possible within the present disclosure as is recognized by the person skilled in the art. According to a preferred embodiment, the reservoir membrane is configured for retaining cells in a filtration process.

[0066] According to a preferred embodiment, the reservoir comprises a reservoir membrane forming a boundary of the reservoir volume, preferably, the reservoir membrane forms a bottom boundary and / or a side boundary of the reservoir volume, more preferably a bottom boundary. By forming a boundary to the reservoir volume, the reservoir membrane is preferably in direct contact with the cell culture liquid, when being inside the reservoir, such that the reservoir membrane can be used to perform a filtration process on the cell culture.

[0067] According to a preferred embodiment, the reservoir membrane is configured for removing and / or adding liquid through the reservoir membrane but essentially retaining the cells within the reservoir volume and / or the reservoir volume and the main volume. Such configuration is particularly advantageous when performing a filtration process throughout culture, as it allows cells to be retained within the reservoir (and optionally casing) but remove liquid. By adding liquid through the membrane, the membrane may be backflushed, e.g. in alternating flow. Additionally, or alternatively, liquid may be added through a supply port, arranged on the retentate side, e.g., in the casing or reservoir.

[0068] According to a preferred embodiment, the reservoir membrane is configured for cultivating the cells in the liquid medium via perfusion in the reservoir volume, and / or the reservoir volume and the main volume; and / or retaining the cells in the reservoir volume when removing a fluid from the reservoir volume and / or the main volume, such as when washing the cells in the reservoir volume, preferably configured for washing the cells in the reservoir volume. Such embodiment is particularly advantageous, as the reservoir membrane allows for performing filtration processes at small scale, e.g. as may be required when low cell numbers are only present, such as for autologous cell therapy. In such an approach, the cells may need to undergo certain process steps, such as activation, transduction and / or transfection. Such process steps may advantageously be performed utilizing the reservoir membrane, wherein in a first step a certain agent may be provided to the reservoir comprising the cells, followed by incubation, followed by removal of liquid through the reservoir membrane. Thereby, the cells are retained, whereas the agent is removed. Afterwards washing steps may be performed and / or addition of cell culture medium for subsequent culture of the cells. Hence, the vessel allows for performing such complex processes inside the same vessel in which the expansion process is performed. Cells do not need to undergo transfer steps, as conventionally required for manufacture of a cell therapy product.

[0069] According to a preferred embodiment, the reservoir comprises a plane bottom, in particular formed at least in part by the reservoir membrane. Adjacent to the membrane in such embodiment may be a support, that supports the membrane. Furthermore, a collection configuration may be provided that allowed for collecting the fluid passing through the membrane and advantageously allowing the fluid to exit, e.g. through a port.

[0070] According to one embodiment, the reservoir comprises a reservoir membrane, preferably wherein the casing does not comprise the reservoir membrane.

[0071] Further vessel features

[0072] According to a preferred embodiment, the vessel is single-use and pre-sterilized such that the casing and the reservoir and the main volume and the reservoir volume are sterile.

[0073] According to another preferred embodiment, the casing comprises at least one sensor element for measuring a parameter or property of the cells and / or the fluid, such as the liquid medium, in the reservoir volume; and / or one or more sensor ports configured for allowing a sensor to be inserted.

[0074] According to a preferred embodiment, the reservoir comprises at least one sensor element for measuring a parameter or property of the cells and / or the fluid, such as the liquid medium, in the reservoir volume; and / or one or more sensor ports configured for allowing a sensor to be inserted. The term "sensor element" is known in the art and particularly refers to a transducer or probe, such as an electrode. The "sensor unit" is known in the art and particularly refers to a transmitter. The sensor element may be connected to the sensor either directly or as a separate unit connected to it. The connection can be also wireless. The sensor unit may be an external sensor unit, for example of the system.

[0075] The sensor(s) of the reservoir and / or casing can be either single-use or multiple use sensors and work as in-line or on-line measurement in direct fluidic connection to the fluid (such as liquid medium) and / or the cells. Further options are process-near at-line or sample based off-line measurements without fluidic connection. The functionality differs with respect to the requested parameter and data between direct or soft-sensors measurement. Due to high sterility requirements single-use sensors are preferred and used in direct monitoring and control strategy of the respective bioprocess.

[0076] The term "sensor port" is known in the art and refers to the accessibility of the sensor to secure the contact of the sensor to the medium and / or cells within the vessel. The sensor port thereby forms a support structure for the sensor elements and may be designed as separate part that can be integrated in the casing and / or reservoir of the vessel by molding, welding and / or as dip tube housing the sensor element.

[0077] According to another embodiment the "sensor port" supports spectroscopy measurements and therefore comprises a flow cell or is designed as spectroscopy port forming an integral part of the casing and / or the reservoir. The flow cell or spectroscopy port can be also located downstream of the cultivation vessel.

[0078] According to a preferred embodiment, the at least one sensor element is arranged upstream of the reservoir membrane, preferably above the reservoir membrane.

[0079] According to a preferred embodiment, the at least one sensor element is arranged at a boundary of the reservoir volume, preferably arranged at a side boundary.

[0080] According to a preferred embodiment, the at least one sensor element is arranged in or at a wall of the reservoir, preferably a side wall of the reservoir, preferably arranged at a side boundary of the reservoir volume. According to a preferred embodiment, the at least one sensor element is arranged at the retentate side of the reservoir volume.

[0081] According to a preferred embodiment, the reservoir is a single molded piece; comprises a material that is optically transparent with a sensor element, in particular a dissolved oxygen sensor element and / or a pH sensor element mounted on the inside of the reservoir and readable through the reservoir; and / or comprises a reservoir wall that is thinned at a location of the sensor element.

[0082] An optical transparent material supports all opto-chemical measurements through the material ad is therefore suitable for pH and dissolved oxygen spots and / or other measurements with optical fibers involved. Sensor spots could be either pre-sterilized and already in place in in the casing or reservoir or protected by a coating that is harmless for the cells.

[0083] According to a preferred embodiment, a further sensor element is arranged downstream of the reservoir membrane, preferably wherein such further sensor element is a flow sensor and / or pressure sensor.

[0084] According to another embodiment the at least one sensor element is based on the measurement principle of an opto-chemical sensor spot, preferably for pH and / or dissolved oxygen; electrochemical electrode, preferably for pH detection; a temperature sensor, preferably a Pt 1000 probe; impedance or capacitance measurement principles, preferably for biomass or viable cell density; and / or all type of spectroscopy principles like UV-Vis absorption, RAMAN, N IR / MIR and / or fluorescence preferably for metabolites or product titer. The sensor elements can be either single-use elements, e.g. sensor spots and / or single-use electrodes, or re-usable, e.g. temperature probes. In another embodiment the measurement principle is based on optical imaging preferably for cell counting, cell viability and / or detection of morphologic cell properties.

[0085] According to a preferred embodiment, the vessel comprises a recirculation loop, preferably a tube loop leading from the vessel and to the vessel. According to a preferred embodiment, the recirculation loop is configured for analysis, including cell counting. For instance, the recirculation loop may comprise a sensor or system for counting cells. According to a preferred embodiment, the recirculation loop, preferably the tube loop, comprises at least one branch. According to a preferred embodiment, the vessel comprises one or more ports, preferably wherein the reservoir comprises at least one port. The one or more ports may be advantageously used to provide access points into and outside of the vessel. In one embodiment, the vessel comprises at least one supply port, at least one waste port, and at least one gas port. According to a particular embodiment, the vessel comprises one or two supply ports, two waste ports and one gas port.

[0086] According to a preferred embodiment, the reservoir is configured to be heated.

[0087] According to a preferred embodiment, the reservoir volume is at most 250 ml, preferably at most 150 ml or 120 ml; and / or the main volume is at most 6 I, preferably at most 3 I or 2 I. Such volumes are particularly suitable for generating a cell therapy product, such as an autologous cell therapy product, wherein typically a low volume cell sample is expanded over time with increasing volume.

[0088] According to a further embodiment, the reservoir volume is at least 1 ml, preferably at least 5 ml, at least 10 ml, at least 15 ml, at least 20 ml, more preferably at least 25 ml or 30 ml. According to a further embodiment, the reservoir volume is selected from the range of 1 ml to 250 ml, preferably 5 ml to 225 ml, 10 ml to 200 ml, 15 ml to 175 ml, 20 ml to 150 ml, more preferably 25 ml to 150 ml or 25 ml to 120 ml.

[0089] According to a further embodiment, the main volume is at least 100 ml, preferably at least 200 ml, at least 300 ml, at least 400 ml, more preferably at least 500 ml. According to a further embodiment, the main volume is selected from the range of 100 ml to 6 I, preferably 200 ml to 5 I, 300 ml to 1, 400 ml to 3.5 I, more preferably 500 ml to 3 I or 500 ml to 2 I.

[0090] According to a preferred embodiment, the vessel comprises a binding unit; and / or is fluidically connected to at least one a binding unit, wherein the binding unit comprises a binding matrix configured for allowing direct or indirect binding of a ligand expressed by at least a fraction of the cells. Such binding unit is particularly suitable for performing cell selection. In particular, by providing a binding unit, at least a fraction of the cells in the liquid medium may be sorted by expressing a ligand that binds to the binding matrix. Cells that do not express such ligand would not bind to the binding matrix. Such binding may be referred to as "direct" binding of the matrix to the ligand expressed in at least a fraction of the cells. Alternatively, one or more binding reagent(s) may be provided, which allows for "indirect" binding of the binding matrix to the ligand expressed in at least a fraction of the cells. In such case, the one or more binding reagent(s) usually bind to the ligand expressed by the cells, e.g. an antibody or antibody fragment capable of binding to the ligand expressed by the cells. The binding reagent then usually further comprises a moiety for binding to the binding matrix, e.g. biotin, avidin, streptavidin, such thatthe cells that express the ligand indirectly (i.e., via the binding reagent) bind to the binding matrix. When providing more than one binding reagent, a first binding reagent may bind to the ligand expressed by the cells, whereas the second binding reagent binds to the binding matrix and both, the first and second binding reagent bind to each other. Further configurations or chemistries may be applied, which achieve the desired outcome, i.e. direct or indirect binding of the cells expressing the ligand to the binding matrix. The skilled person is well-aware of suitable binding matrices and chemistries for allowing binding of a ligand expressed by at least a fraction of the cells.

[0091] The vessel may be fluidically connected to the vessel, wherein such fluidic connection may be provided in form of tubes. There may be more than one fluidic connection, such as preferably two fluidic connections, in particular at different sides or positions of the binding unit. For instance, there may be one fluidic connection from the vessel to the entrance of the binding unit and one fluidic connection from the exit of the binding unit to the vessel. Further fluidic connections may be present, which allow also fluid to be transferred through the binding unit into another container, e.g. waste container or storage container. The fluidic connection(s) between the vessel and the binding unit may be located at the casing and / or the reservoir of the vessel. Preferably, there is one fluidic connection between the reservoir and the binding unit and another fluidic connection between the casing and the binding unit. Other configurations are also possible, for instance, there may be two fluidic connections between the casing and the binding unit. Or, alternatively, two fluidic connections between the reservoir and the binding unit.

[0092] After binding of the at least fraction of the cells, liquid medium present in the vessel can be removed, e.g. through the binding matrix or a fluidic line connected to vessel, and with the liquid the unbound cells (which then do not express the ligand). As a result, only cells expressing the ligand would remain in the vessel, i.e., a positive cell selection is performed. In order to release the cells from the binding matrix, these may be eluted as commonly known in the art (e.g. by providing a release agent, such as salt, biotin, avidin or streptavidin). Eluted cells would then be released into the liquid medium present in the vessel. Preferably, the cells can be recirculated into the vessel, e.g. by pressing liquid through the binding unit which elutes the cells and then into a fluidic line connected to the vessel, such that the released cells recirculate into the vessel. Afterwards, the binding unit may be closed or becomes inactive. The binding unit may also still be left "open", so in principle being accessible to the cells. However, no force or pressure is preferably applied towards the binding unit, such that the cells are not actively moved into the binding unit. The binding unit may also be closed, e.g., by closing a valve or a cover that closes the binding unit from the vessel.

[0093] Also, a negative cell selection step may be performed. For instance, after binding of the at least fraction of the cells, liquid medium present in the vessel can be removed, e.g. through the binding matrix or a fluidic line connected to vessel, and with the liquid the unbound cells (which then do not express the ligand). These unbound cells can then be collected, e.g. in a container. In order to release the cells from the binding matrix, these may be eluted as commonly known in the art. Eluted cells would then be released into the liquid medium present in the vessel and can then be removed through a waste port. Preferably, the cells can be released from the binding unit, e.g. by pressing liquid through the binding unit which elutes the cells and then into a fluidic line connected to a waste container, such that the released cells are removed from the vessel. Afterwards, the collected cells may be recirculated back to the vessel.

[0094] According to some embodiments of the present disclosure more than one cell selection step is performed, such as at least two cell selection steps. These may comprise positive and / or negative cell selection, as disclosed herein.

[0095] According to a preferred embodiment, the binding matrix is provided as a resin or monolithic material. Various materials are known in the art for binding matrices. Particularly preferred may be a binding matrix which comprises resin beads. A binding matrix may be covered or enclosed by one or more membranes, which separates the cells from the binding matrix, as long as no force is applied that would drag the cells in contact with the binding matrix.

[0096] According to a preferred embodiment, the binding unit is located within or adjacent to the reservoir of the vessel, preferably wherein the binding unit is arranged adjacent to the reservoir membrane. By providing the binding unit located within or adjacent to the reservoir, small volumes of cell culture or cell suspension can be handled. Specifically, the cells may be provided as a mixture of cells, which need to undergo a cell selection step utilizing the binding matrix. Since the cells are in such stage oftentimes present in low volume cell cultures, it is advantageous to provide the binding unit in the reservoir of the vessel, which can be advantageously used to handle and culture low volume cell cultures. I n addition, the reservoir is pre-shaped, such that it is mechanically more stable than a reservoir that only shapes upon interaction with other devices throughout culture. Thus, the binding unit has then a fixed position, allowing for precise cell selection. In the embodiments, wherein the binding unit is located within or adjacent to the reservoir of the vessel, the binding unit may be provided as a column, wherein the column is predominantly present internally, i.e., within the vessel, particularly within the reservoir, such that the column reaches into the reservoir volume. Such "internal column" configuration advantageously allows for the cells present in the cell vessel and reservoir, respectively, to be in direct connect with the column, such that no additional fluid transfer step is required. In such configuration, it may be advantageous to provide the binding matrix within the column covered by a membrane, such that the cells are not always in contact with the binding matrix in order to control the cell selection. As a result, the cells only interact with the binding matrix, once a force is put on the cells that drags them through the membrane into the column and thus in contact with the binding matrix. As a result, the cell selection is only occurring when such force is applied. According to one embodiment, an active element, e.g. a pump, is provided that manipulates the fluid in the vessel such that the fluid is dragged into the column. For instance, a pump may draw the fluid and with it the cells present in the fluid within the vessel into the column, such that the cells come in contact with the binding matrix, such that at least a fraction of the cells bind to the binding matrix. According to one embodiment, the binding unit further comprises a valve, which is configured for closing the fluid lines exiting the binding unit. This advantageously closes the binding matrix, such that cells are not dragged actively into the binding unit and this in contact with the binding matrix.

[0097] According to one preferred embodiment, the binding unit is provided as a column, preferably the opening of the column is arranged within an opening of the reservoir, more preferably an opening at the bottom of the reservoir. This has the advantage that the column can be used as known in the art for cell selection columns, however, as implementation of the cell vessel, such that cell selection can be performed with the same vessel, in which cell culture can take place, as well as preferably cell activation, cell transduction, cell transfection, media exchange, and / or volume reduction. Hence, relevant steps for manufacture of a cell therapy product can be performed within the same vessel, significantly simplifying the process.

[0098] Additionally or alternatively, the binding unit is provided as a column which is fluidically connected to the vessel, preferably the reservoir, wherein fluidic connections between the vessel and the column are provided, in particular wherein the fluidic connections are provided by one or more tubes, optionally further comprising a valve. Fluidically connecting the column and the reservoir has the advantage that the binding unit is separate from the vessel allowing for separate control. At the same time both are usually in close proximity, such that for instance a temperature control would effectively heat the vessel and the column, which is advantageous for maintaining high cell viability. Optionally, the fluidic connection, such as one or more tubes further comprise a valve. This has the advantage that the fluidic connection between the binding unit (in particular column) and the vessel (in particular reservoir) can be closed, which can be done subsequent to the cell selection.

[0099] Providing the binding unit as a column as disclosed herein the term "column" shall not be limited in any specific geometry, as is known in the art. While typically columns may be provided in a pillar or column shape, also other shapes are known in the field. A column herein may also refer to a bag-like shape, wherein two or more walls are connected forming a casing. As typically casings who have a bag-like shape are flexible, such column configuration may also be referred to as "flexible column".

[0100] According to a preferred embodiment, the binding unit comprises a flexible column, comprising a casing, an entrance port and an exit port, wherein the casing comprises the binding matrix. The flexible column may further comprise two membranes, which confine the casing volume in which the binding matrix is present, e.g. one membrane at the entrance and one membrane at the exit of the flexible column. The flexible column may be fluidically connected to the vessel, wherein preferably such connection comprises valve to controlling the fluid flow between the vessel and the flexible column.

[0101] According to another preferred embodiment, the binding unit is provided as a floating unit configured for float within the reservoir volume and / or the main volume. When providing the binding unit as a floating unit, this may be provided in form of a pouch, which can float within a fluid of the vessel. Such floating unit is typically connected via fluidic line(s) going through the vessel to the outside. Hence, the floating unit can float but within a certain range limited to the length of the fluidic line(s). A floating unit has the advantage that it is present within the vessel, such that contact between the floating unit and the cells within the vessel is enabled. In order to achieve interaction between the cells and the binding matrix of the binding unit, the cells may be forced or pushed into the floating binding unit by overpressure within the vessel and / or under pressure within the binding unit, e.g. created by the fluidic line(s). Bound cells can then, as explained above, be retained within the binding matrix, whereas unbound cells exit the binding unit through the fluidic line(s).

[0102] According to a preferred embodiment, the binding unit is by an external loop fluidically connected to the vessel, particularly the reservoir, and / or a waste port, preferably both. A specific configuration of such embodiment can be seen in Fig. 9. By providing an external loop it is possible to recirculate the cells though the binding unit into the vessel, particularly the reservoir, and thus the vessel again. This has the advantage that cells can undergo multiple rounds of binding to achieve binding of as many cells as possible expressing the ligand that binds to the binding matrix. Furthermore, this has the advantage that cells that bind to the binding matrix and are subsequently eluted, can exit the binding unit and be recirculated into the cell vessel, in particular into the reservoir. Furthermore, by connecting the binding unit to a waste port, it is possible to transfer the unbound cells into a waste container.

[0103] According to a preferred embodiment, the external loop comprises at least one valve, preferably one valve for controlling the fluid flow between the binding unit and the waste port and another valve controlled the fluid flow between the vessel, e.g. reservoir, and the waste port, preferably wherein both valves are for controlling the fluid flow between the binding unit and the vessel, e.g. reservoir, via the external loop. A specific configuration of such embodiment can be seen in Fig. 9. By providing the valves, the fluid flow can be controlled, allowing for fluid flow into a waste container and / or recirculation into the vessel, particularly into the reservoir.

[0104] System for cultivating cells in a liquid medium in a cultivation procedure according to a second aspect of the invention

[0105] According to a second aspect of the invention, a system for cultivating cells in a liquid medium in a cultivation procedure is provided, wherein the system comprises a vessel according to the first aspect of the invention.

[0106] The system according to the second aspect advantageously allows for cultivating cells from small to large scale. Specifically, the system utilizes the advantages as outlined above for the vessel according to the first aspect of the invention, including simplified and efficient cell expansion, but also as is demonstrated in the Examples below, higher daily expansion rates and cell viabilities can be achieved, such that cultivation duration can be shortened and product quality is im roved (see e.g., Figs. 7 and 8).

[0107] The individual features and preferred embodiments of the system according to the second aspect mostly correspond to the individual features and embodiments of the vessel according to the first aspect. Therefore, it is referred to the above disclosure which shall equally be applicable for the system according to the second aspect. This particularly but not exclusively includes the casing, the filter, including the main membrane and reservoir membrane, the sensor, and further vessel features, such as the recirculation loop and binding unit. Further features will now be described in detail.

[0108] The ''system" may also be referred to a ''bioprocessing system" and is in particular suitable for cultivating cells in a liquid medium in a cultivation procedure. It may also be suitable for performing a bioprocess on a cell culture.

[0109] According to a preferred embodiment, the system further comprises: i. a rocker platform, preferably wherein the vessel is attached to the rocker platform, more preferably such that the rocker platform can pivot the vessel; ii. a sensor unit, preferably wherein the sensor unit is connected to the vessel via the sensor unit connector; and / or

[0110] Hi. a heating arrangement, preferably wherein the vessel is heated by the heating arrangement, in particular such that the surrounding area of the vessel is heated.

[0111] According to a preferred embodiment, the sensor unit is connected to the vessel in an assembly movement of the vessel, in which the vessel is attached to the rocker platform.

[0112] According to a preferred embodiment, the heating arrangement is configured for heating a casing part, preferably a lower casing part and an upper casing part, and / or the reservoir.

[0113] The system may comprise: i. at least one exchangeable bioreactor module comprising a cell vessel area, wherein in the cell vessel area a cell vessel can be located, and a media supply area, wherein in the media supply area a media container can be located; ii. a framework, and iii. a transport mechanism for automatically transporting the bioreactor module.

[0114] The "cell vessel" in such embodiment advantageously corresponds to the vessel according to the first aspect of the invention. Such system advantageously allows for simplified handling of the at least one exchangeable bioreactor module (and thus the vessel). By automatically transporting the bioreactor module the bioprocessing system advantageously allows to transport the bioreactor module between different positions for performing a bioprocess without a requirement for the user or operator to interfere. For simplified handling, the cell culture media supply is transported together with the cell vessel, wherein the media is replenished, either by refilling the media container or by adding a new media container. As such, the complexity and size of the bioreactor module are reduced. If the medium needs certain storage conditions, it may be possible to refill the media container often enough to eliminate the need for providing these storage conditions at the media supply area. The combination of having a media container and replenishing the medium provides for a less complex transport mechanism and allows functional grouping in the bioprocessing system.

[0115] The system according to the present disclosure may comprise a transport mechanism for automatically transporting the bioreactor module. The "transport mechanism" may also be referred as a transport unit. It advantageously allows for automatically transporting the bioreactor module between different positions. Preferably, the bioreactor module comprises the cell vessel and the media container during the automated transportation. As a result, the different system features required for cell culture are handled together as a single unit which can be handled by the transport mechanism of the system. Indeed, the present disclosure has the object to provide more than one bioreactor module which can be consecutively (or simultaneously) transported in an automated manner using the transport mechanism disclosed herein. For instance, in Fig. 1 it is further the case that the system comprises a transport mechanism, for example a movable tray mounted on rails (located outside the visible area on the right side in Fig. 1) or a robotic arm, for automatically moving the bioreactor module. The transport mechanism may transport the cell vessel and the media container at the same time and together as both are connected fluidically.

[0116] According to further embodiments, the transport mechanism is configured to automatically transfer the bioreactor module between different positions, preferably between an expansion location and at least one further position, which can be the refill location, the emptying location, and / or the swapping location. At the different positions, specific bioprocessing steps can be performed such that the functions of different positions within the bioprocessing system can be optimized for the respective purpose. For instance, the transport mechanism may transport the bioreactor module in an automated manner to the refill location, the emptying location, and / or the swapping location, all of which may be the same or different positions. In one preferred embodiment, the refill location, the emptying location, and the swapping location correspond to a single position. At this position, the bioreactor module may be equipped with a suitable consumable, e.g. including the cell vessel, as well as a media container and waste container (or alternatively the life support container). In addition, different bioprocessing steps may be performed, including inoculation of the cell vessel, activation and transduction, but optionally, also filling of the media container with fresh cell culture media. Afterwards, the bioreactor unit may be transported by the transport mechanism to another location, such as the expansion location, whereat the cell culture undergoes expansion. In case during expansion the media container does not contain a sufficient volume for the whole culture, the me ia container may be transported to the refill location, the emptying location, and / or the swapping location by the transport mechanism. Here, the media container may be swapped with a new media container (automatically or manually), or the media container may be connected to a media supply container and fresh media may be filled into the media container, essentially "refilling" the container. Similarly, the waste container may be swapped or emptied in case throughout culture the waste container volume is insufficient to store the complete permeate / waste volume.

[0117] The transport mechanism can be an elevator, such that the bioreactor module can be elevated into different positions in respect to the height, e.g. allowing for moving the bioreactor module into different positions provided by the framework of the bioreactor module.

[0118] According to further embodiments, the bioreactor module further com prises a support structure, wherein due to the support structure the bioreactor module can be handled as a unit. In particular, due to the support structure the bioreactor module can be automatically transported by the transport mechanism. Preferably, due to the support structure the bioreactor module can be handled as a unit together with the cell vessel, the media container and, optionally, a waste container.

[0119] According to further embodiments, the support structure of the bioreactor module comprises a life support area. In the life support area, a life support container can be placed. The life support container may also be placed in the life support area by being mounted on or placed on a life support tray. As disclosed herein, the life support container or life support tray can be located "in" the life support area. Specifically, this means that the life support container or life support tray can be located "at" or "on" the life support area, as is well understood by the person skilled in the art.

[0120] Providing a life support container, e.g. mounted on a life support tray, has the advantage the advantage that it can be handled as a single piece. Such life support container advantageously comprises a media container and a waste container, such that the media container and waste container do not need to be placed at different locations and handled as two individual objects. Rather, one single object, i.e. the life support container, can be handled. It may also be within the scope of the present disclosure that further containers are provided by the life support container, which may also be mounted or placed on the life support tray. In case a life support area is provided, preferably the media supply area would essentially overlap with the life support area. Preferably, also the waste area would essentially overlap with the life support area.

[0121] Hence, according to further embodiments, the system further comprises a cell vessel located in the cell vessel area, wherein the cell vessel is a vessel according to the first aspect of the invention, a media container located in the media supply area, optionally a waste container located in a waste area.

[0122] Preferably in such embodiments, the bioreactor module may not comprise a life support area. Alternatively, the bioreactor module comprises a life support area, which comprises the media supply area and the waste area.

[0123] According to further embodiments, the system further comprises a cell vessel located in the cell vessel area, wherein the cell vessel is a vessel according to the first aspect of the invention, a life support container located in a life support area.

[0124] Preferably, in such embodiment, the life support area overlaps or comprises the media supply area.

[0125] According to further embodiments, the system comprises a life support area. Preferably, the life support area comprises the media supply area and / or the waste area, preferably wherein the media supply area is the same area as or a different area than the waste area on the life support area, more preferably wherein the media supply area is the same area as the waste area.

[0126] According to further embodiments, the life support area comprises the media supply area and / or the waste area such that the life support container contains a media container and / or a waste container.

[0127] According to further embodiments, the support structure further comprises a rocking unit for rocking the cell vessel. The rocking unit is advantageous in order to perform a rocking motion or wave like cultivation of the cells in the cell vessel. This allows to provide adequate gas exchange through the gas-liquid interface in the cell vessel without the need for further oxygenation, e.g. via a sparger. However, an external additional gas supply may also be provided, e.g. in form of a sparger, if needed. According to further embodiments, the support structure further comprises a balance for weighing a life support tray. Weighing the support try has the advantage that the containers placed on the tray can be closely tracked, such that the incoming and exiting volume can be tracked. According to a preferred embodiment, the balance weighs the life support tray: before a transfer of a liquid medium from the media supply area to the cell vessel, after a transfer of a liquid medium from the media supply area to the cell vessel, during a transfer of a liquid medium from the media supply area to the cell vessel, before a transfer of a liquid from the cell vessel to the waste area, after a transfer of a liquid from the cell vessel to the waste area, and / or during a transfer of a liquid from the cell vessel to the waste area to determine and / or control an amount of transferred liquid medium. Liquid medium may encompass any liquid, including cell culture media, waste liquid but also wash liquid or liquid comprising a reagent.

[0128] According to further embodiments, the support structure further comprises a balance for weighing a media container. Hence, the balance may weigh the media container: before a transfer of a liquid medium from the media supply area to the cell vessel, after a transfer of a liquid medium from the media supply area to the cell vessel, and / or during a transfer of a liquid medium from the media supply area to the cell vessel to determine and / or control an amount of transferred liquid medium. Liquid may encompass cell culture media liquid.

[0129] According to further embodiments, the support structure further comprises a balance for weighing a waste container. Hence, the balance may weigh the waste container: before a transfer of a liquid from the cell vessel to the waste area, after a transfer of a liquid from the cell vessel to the waste area, and / or during a transfer of a liquid from the cell vessel to the waste area to determine and / or control an amount of transferred liquid medium. Liquid may encompass waste liquid.

[0130] In a further embodiment, the support structure further comprises a balance for weighing a cell vessel. For instance, the balance may be located at the cell vessel area, such that the cell vessel may be weighed during any liquid transfer to determine and / or control an amount of transferred liquid medium. According to further embodiments, the system is configured for performing one or both of the following operations in an automated manner: a refill routine, preferably wherein the system automatically connects a refill container comprising a liquid medium to the media container and automatically refills the media container with the liquid medium and preferably automatically disconnects the refill container from the media container, optionally during a cell culture cycle; and an emptying routine, preferably wherein the bioprocessing system automatically connects a further waste container to the waste container and automatically empties the waste container from the waste and preferably automatically disconnects the further waste container from the waste container, optionally during a cell culture cycle.

[0131] According to further embodiments, the system, preferably the framework of the system, comprises a refill location, at which a refill routine can be performed; an emptying location, at which an emptying routine can be performed; and / or a swapping location, at which a swapping operation can be performed, preferably wherein the system performs the swapping and / or at the swapping location the swapping is performed manually. optionally, wherein the system preferably the framework of the bioprocessing system, comprises the refill location and the emptying location, both of which have the same location.

[0132] According to further embodiments, the system, preferably the framework of the system, comprises at least one expansion location, preferably several expansion locations.

[0133] According to further embodiments, the framework of the system comprises several expansion locations, wherein the expansion locations are placed vertically, more preferably, that the expansion locations are compartments in a shelf with one or more vertical columns.

[0134] According to further embodiments, the bioprocess comprises expanding the cells or the cell culture, preferably wherein expanding the cells is performed predominantly at an expansion location of the system.

[0135] According to further embodiments, the cell expansion location(s) is / are outside a swapping location, an emptying location and / or a swapping location. According to further embodiments, the system, preferably the framework of the system, comprises a connection arrangement configured for aseptically connecting and / or disconnecting one or more, preferably all, of the following the media container and the cell vessel, the media container and the refill container, and the waste container and the cell vessel, preferably, that the connection arrangement comprises a tube welding unit for welding together and / or cutting and closing off one or more tubes connected to the media container and the cell vessel, and preferably the waste container and the cell vessel, preferably in an automated manner. The welding unit may employ thermal, ultrasonic, or other suitable welding techniques to ensure a secure and sterile seal. To verify the integrity of the sealed connection, the system may further include a seal testing unit capable of assessing whether the sealing process was successful. This testing may be performed by applying pressure, vacuum, or other diagnostic methods to detect leaks or structural weaknesses in the sealed region.

[0136] According to further embodiments, the connection arrangement is movable, preferably movable in an automated manner, wherein the connection arrangement can be moved to be located at the swapping location, at the refill location, and / or at the emptying location. By being movable, the connection arrangement, e.g. tube welder can advantageously move the respective positions in order to perform a connection and / or disconnection.

[0137] According to further embodiments, the bioreactor module, in particular the life support tray, comprises at least one tube holder configured for holding at least one tube, preferably for holding at least one tube in a defined position relative to the support structure and / or the life support container.

[0138] According to further embodiments, the tube holder comprises a tube mover mechanism for extending and / or retracting the at least one tube relative to the tube holder.

[0139] According to further embodiments, a connection arrangement, preferably a tube welding unit, interacts with the tube holder to weld and / or cut and seal the at least one tube.

[0140] According to further embodiments, the bioreactor module further comprises a lid for providing a controlled environment for the cell vessel; and / or a heating unit for the cell vessel. Method for cultivating cells in a liquid medium according to a third aspect of the invention

[0141] According to a third aspect of the invention, a method for cultivating cells in a liquid medium is provided, comprising: a) providing cells in a liquid medium; b) cultivating the cells using a vessel according to the first aspect of the invention and / or a system according to the second aspect of the invention.

[0142] The method according to the third aspect advantageously allows for cultivating cells from small to large scale. Specifically, the method utilizes the advantages as outlined above for the vessel according to the first aspect of the invention and / or the system according to the second aspect of the invention, including simplified and efficient cell expansion, but also as is demonstrated in the Examples below, higher daily expansion rates and cell viabilities can be achieved, such that cultivation duration can be shortened and product quality is improved (see e.g., Figs. 7 and 8).

[0143] The individual features and preferred embodiments of the method according to the third aspect mostly correspond to the individual features and embodiments of the vessel accor ing to the first aspect and to the system according to the second aspect. Therefore, it is referred to the above disclosure which shall equally be applicable for the method according to the third aspect. This particularly but not exclusively includes the casing, the filter, including the main membrane and reservoir membrane, the sensor, and further vessel features, such as the recirculation loop and binding unit, as well as the system features, including the framework, bioreactor module, refilling and emptying. Further features will now be described in detail.

[0144] According to a preferred embodiment, the cells are dedicated for cell and / or gene therapy, preferably dedicated for cell therapy.

[0145] According to a preferred embodiment, the cells are immune cells, preferably T cells.

[0146] According to a preferred embodiment, the cells are genetically modified T cells, preferably genetically modified CAR-T cells.

[0147] According to a preferred embodiment, the cells become genetically modified, preferably, wherein the method comprises providing an agent for genetically modifying the cells present in the vessel, more preferably wherein the cells are T cells. According to a preferred embodiment, step (b) comprises

[0148] (b. l) a preliminary cultivation phase, in which the cells are preliminarily cultivated, preferably wherein cells are predominantly cultivated in the reservoir; and

[0149] (b.2) a subsequent cultivation phase, in which the cells are subsequently cultivated preferably wherein cells are cultivated in the reservoir and the casing.

[0150] According to a preferred embodiment, the reservoir defines a reservoir volume for receiving the liquid medium in the preliminary cultivation phase, wherein the casing defines a main volume for receiving the liquid medium in the subsequent cultivation phase.

[0151] According to a preferred embodiment, the method further comprises one or more of the following steps: optionally, selecting cells one or more times, in particular by binding at least a fraction of the cells expressing a ligand, preferably selecting a subpopulation of T cells; activating cells, preferably activating T cells; transducing cells, preferably transducing T cells; transfecting cells, preferably transfecting T cells; and / or expanding cells, preferably T cells, more preferably genetically modified T cells; preferably, wherein the method comprises at least two or three of the above-mentioned steps.

[0152] According to a preferred embodiment, the method comprises after step a) but before step b): optionally, selecting cells one or more times, preferably by binding at least a fraction of the cells expressing a ligand; activating cells, preferably activating T cells; and transducing cells, preferably transducing T cells, or transfecting cells, preferably transfecting T cells.

[0153] Method for manufacturing a cell therapy product according to a fourth aspect of the invention

[0154] According to a fourth aspect of the invention, a method for manufacturing a cell therapy product is provided comprising: a) providing cells in a liquid medium; b) cultivating the cells using a vessel according to the first aspect of the invention and / or a system according to the second aspect of the invention. The method according to the fourth aspect advantageously allows for manufacturing a cell therapy product, including cultivating cells from small to large scale. Specifically, the method utilizes the advantages as outlined above for the vessel according to the first aspect of the invention and / or the system according to the second aspect of the invention, including simplified and efficient cell expansion, but also as is demonstrated in the Examples below, higher daily expansion rates and cell viabilities can be achieved, such that cultivation duration can be shortened and product quality is improved (see e.g., Figs. 7 and 8).

[0155] The individual features and preferred embodiments of the method according to the fourth aspect mostly correspond to the individual features and embodiments of the vessel according to the first aspect and to the system according to the second aspect. Therefore, it is referred to the above disclosure which shall equally be applicable for the method according to the fourth aspect. This particularly but not exclusively includes the casing, the filter, including the main membrane and reservoir membrane, the sensor, and further vessel features, such as the recirculation loop and binding unit, as well as the system features, including the framework, bioreactor module, refilling and emptying. Further features will now be described in detail.

[0156] According to a preferred embodiment, the cells are immune cells, preferably T cells.

[0157] According to a preferred embodiment, herein the cells are genetically modified T cells, preferably genetically modified CAR-T cells.

[0158] According to a preferred embodiment, the method further comprises one or more of the following steps: optionally, selecting cells one or more times, in particular by binding at least a fraction of the cells expressing a ligand, preferably selecting a subpopulation of T cells; activating cells, preferably activating T cells; transducing cells, preferably transducing T cells; transfecting cells, preferably transfecting T cells; and / or expanding cells, preferably T cells, more preferably genetically modified T cells; preferably, wherein the method comprises at least two or three of the above-mentioned steps.

[0159] According to a preferred embodiment, the method comprises after step a) but before step b): optionally, selecting cells one or more times, preferably by binding at least a fraction of the cells expressing a ligand; activating cells, preferably activating T cells; and transducing cells, preferably transducing T cells, or transfecting cells, preferably transfecting T cells.

[0160] According to a preferred embodiment, the method further comprises after step b) formulating the expanded cell culture into a media suitable for being injected into a patient and / or transferring the expanded cell culture from the cell vessel (6) into a harvesting vessel.

[0161] Uses according to a fifth and sixth aspect of the invention

[0162] According to a fifth aspect of the invention, use of the vessel according to the first aspect of the invention and / or system according to the second aspect of the invention for manufacturing a cell therapy product is provided. Preferably wherein manufacturing comprises at least one of the following: optionally, selecting cells one or more times, in particular by binding at least a fraction of the cells expressing a ligand, preferably selecting a subpopulation of T cells; activating cells, preferably activating T cells; transducing cells, preferably transducing T cells; transfecting cells, preferably transfecting T cells; and / or expanding cells, preferably T cells, more preferably genetically modified T cells.

[0163] According to a sixth aspect of the invention, use of the vessel according to the first aspect of the invention and / or system according to the second aspect of the invention in one of the methods disclosed herein is provided.

[0164] The uses according to the fifth and sixth aspect advantageously allows for manufacturing a cell therapy product, including cultivating cells from small to large scale. Specifically, the uses utilize the advantages as outlined above for the vessel according to the first aspect of the invention and / or the system according to the second aspect of the invention, as well as the methods according to the third and fourth aspects, including simplified and efficient cell expansion, but also as is demonstrated in the Examples below, higher daily expansion rates and cell viabilities can be achieved, such that cultivation duration can be shortened and product quality is improved (see e.g., Figs. 7 and 8).

[0165] The individual features and preferred embodiments of the uses according to the fifth and sixth aspects mostly correspond to the individual features and embodiments of the vessel according to the first aspect and to the system according to the second aspect, as well as according to the methods according to the third and fourth aspect. Therefore, it is referred to the above disclosure which shall equally be applicable for the uses. This particularly but not exclusively includes the casing, the filter, including the main membrane and reservoir membrane, the sensor, and further vessel features, such as the recirculation loop and binding unit, as well as the system features, including the framework, bioreactor module, refilling and emptying.

[0166] Further aspects of the invention

[0167] According to a further aspect of the invention, cultivated cells are provided obtained by the method according to the third aspect of the invention.

[0168] According to a further aspect of the invention, a cell therapy product is provided obtained by the method according to the fourth aspect of the invention.

[0169] The individual features and preferred embodiments of the cultivated cells and cell therapy product according to the further aspects mostly correspond to the individual features and embodiments of the vessel according to the first aspect and to the system according to the second aspect, as well as according to the methods according to the third and fourth aspect. Therefore, it is referred to the above disclosure which shall equally be applicable for these aspects. This particularly but not exclusively includes the casing, the filter, including the main membrane and reservoir membrane, the sensor, and further vessel features, such as the recirculation loop and binding unit, as well as the system features, including the framework, bioreactor module, refilling and emptying.

[0170] Further embodiments of the present disclosure

[0171] The following embodiments provide further advantages of the present disclosure:

[0172] 1. A vessel for cultivating cells in a liquid medium in a cultivation procedure, wherein the vessel (1) comprises a casing (4) defining a main volume (7), and a reservoir (5) defining a reservoir volume (6), wherein the reservoir volume (6) and the main volume (7) are adjacently arranged and are configured to be fluidically connected for fluidic exchange, characterized in that the reservoir (5) is a pre-shaped reservoir (5). The vessel according to embodiment 1, wherein the casing (4) comprises a casing part, preferably two or more casing parts, such as an upper casing part (36) and a lower casing part (37). The vessel according to embodiment 1 or 2, wherein the reservoir (5) is attached to the casing (4), preferably to a casing part, more preferably a lower casing part (37), or the reservoir (5) is formed integrally with the casing (4), preferably with a casing part, more preferably with a lower casing part (37). The vessel according to one or more of embodiments 1 to 3, wherein the vessel (1) comprises at least one filter; and / or is fluidically connected to at least one filter, preferably, wherein the filter is configured to allow liquid to flow through but essentially retain cells. The vessel according to embodiment 4, wherein the at least one filter comprises a filter medium selected from a surface filter and a depth filter. The vessel according to embodiment 4 or 5, wherein the at least one filter is a membrane, such as a planar membrane, a hollow fiber membrane and / or floating membrane. The vessel according to one or more of embodiments 4 to 6, wherein the at least one filter comprises a main membrane (40), and / or a reservoir membrane (42); preferably both of a main membrane (40) and a reservoir membrane (42). The vessel according to embodiment 7, wherein the main membrane (40) is arranged at the casing (4), preferably at a casing part, more preferably at the lower casing part (37); and / or is fluidically connected to the casing (4), preferably wherein fluidic connections between the casing (4) and the main membrane (40) are provided, in particular wherein the fluidic connections are provided by one or more tubes. The vessel according to embodiment 7 or 8, wherein the main membrane (40) surrounds the reservoir, is adjacent to the reservoir (5) and / or floats within the main volume (7), preferably wherein the main membrane is adjacent to the reservoir. The vessel according to one or more of embodiments 7 to 9, wherein the main membrane (40) is configured for removing and / or adding liquid through the main membrane (40) but essentially retaining the cells within the main volume (7) or preferably, within the main volume (7) and the reservoir volume (6). 11. The vessel according to one or more of embodiments 7 to 10, wherein the main membrane (40) is configured for cultivating the cells in the liquid medium via perfusion in the main volume (7) or preferably, in the main volume (7) and the reservoir volume (6).

[0173] 12. The vessel according to one or more of embodiments 7 to 11, wherein the reservoir membrane (42) is arranged at the reservoir (5), preferably the reservoir (5) comprises the reservoir membrane (42), and / or is fluidically connected to the reservoir (5), preferably wherein fluidic connections between the reservoir (5) and the membrane are provided, more preferably wherein the fluidic connections are provided by one or more tubes.

[0174] 13. The vessel according to one or more of embodiments 7 to 12, wherein the reservoir (5) comprises a reservoir membrane (42) forming a boundary of the reservoir volume (6), preferably, the reservoir membrane (42) forms a bottom boundary and / or a side boundary of the reservoir volume (6), more preferably a bottom boundary.

[0175] 14. The vessel according to one or more of embodiments 7 to 13, wherein the reservoir membrane (42) is configured for removing and / or adding liquid through the reservoir membrane (42) but essentially retaining the cells within the reservoir volume (6) and / or the reservoir volume (6) and the main volume (7).

[0176] 15. The vessel according to embodiment 14, wherein the reservoir membrane (42) is configured for cultivating the cells in the liquid medium via perfusion in the reservoir volume (6), and / or the reservoir volume (6) and the main volume (7); and / or retaining the cells in the reservoir volume (6) when removing a fluid from the reservoir volume (6) and / or the main volume (7), such as when washing the cells in the reservoir volume (6), preferably configured for washing the cells in the reservoir volume (6).

[0177] 16. The vessel according to one or more of embodiments 1 to 15, wherein the reservoir (5) comprises a plane bottom, in particular formed at least in part by the reservoir membrane (42).

[0178] 17. The vessel according to one or more of embodiments 1 to 16, wherein the vessel (1) is single-use and pre-sterilized such that the casing (4) and the reservoir (5) and the main volume (7) and the reservoir volume (6) are sterile.

[0179] 18. The vessel according to one or more of embodiments 1 to 17, wherein the reservoir (5) comprises at least one sensor element (16) for measuring at least one parameter of the cells and / or the fluid, such as the liquid medium, in the reservoir volume (6); and / or one or more sensor ports configured for allowing a sensor to be inserted. 19. The vessel according to embodiment 18, wherein the at least one sensor element (16) is arranged upstream of the reservoir membrane (42), preferably above the reservoir membrane (42).

[0180] 20. The vessel according to embodiment 18 or 19, wherein the at least one sensor element (16) is arranged at a boundary of the reservoir volume (6), preferably arranged at a side boundary.

[0181] 21. The vessel according to one or more of embodiments 18 to 20, wherein the at least one sensor element (16) is arranged in or at a wall of the reservoir, preferably a side wall of the reservoir (5), preferably arranged at a side boundary of the reservoir volume (6).

[0182] 22. The vessel according to one or more of embodiments 18 to 21, wherein the at least one sensor element (16) is arranged at the retentate side of the reservoir volume.

[0183] 23. The vessel according to one or more of embodiments 18 to 22, wherein the reservoir (5) is a single molded piece; comprises a material that is optically transparent with a sensor element (16), in particular a dissolved oxygen sensor element (16) and / or a pH sensor element (16) mounted on the inside of the reservoir (5) and readable through the reservoir (5); and / or comprises a reservoir wall (46) that is thinned at a location of the sensor element (16).

[0184] 24. The vessel according to one or more of embodiments 18 to 23, wherein a further sensor element is arranged downstream of the reservoir membrane (42), preferably wherein such further sensor element is a flow sensor and / or pressure sensor.

[0185] 25. The vessel according to one or more of embodiments 1 to 24, wherein the vessel (1) comprises a recirculation loop, preferably a tube loop (49) leading from the vessel (1) and to the vessel (1).

[0186] 26. The vessel according to embodiment 25, wherein the recirculation loop is configured for analysis, including cell counting.

[0187] 27. The vessel according to embodiment 25 or 26, wherein the recirculation loop, preferably the tube loop (49), comprises at least one branch (50).

[0188] 28. The vessel according to one or more of embodiments 1 to 27, wherein the vessel (1) comprises one or more ports, preferably wherein the reservoir comprises at least one port.

[0189] 29. The vessel according to one or more of embodiments 1 to 28, wherein the reservoir is configured to be heated.

[0190] 30. The vessel according to one or more of embodiments 1 to 29, wherein the reservoir volume is at most 250 ml, preferably at most 150 ml or 120 ml; and / or the main volume is at most 6 I, preferably at most 3 I or 2 I.

[0191] 31. The vessel according to one or more of embodiments 1 to 30, wherein the vessel (1) comprises a binding unit (101); and / or is fluidically connected to at least one a binding unit (101), wherein the binding unit (101) comprises a binding matrix configured for allowing direct or indirect binding of a ligand expressed by at least a fraction of the cells.

[0192] 32. The vessel according to embodiment 31, wherein the binding matrix is provided as a resin or monolithic material.

[0193] 33. The vessel according to embodiment 31 or 32, wherein the binding unit (101) is located within or adjacent to the reservoir (5) of the vessel (1), preferably wherein the binding unit is arranged adjacent to the reservoir membrane (42).

[0194] 34. The vessel according to one or more of embodiments 31 to 33, wherein the binding unit (101) is provided as a column, preferably wherein the opening of the column is arranged within an opening of the reservoir (5), more preferably an opening at the bottom of the reservoir (5), and / or the column is fluidically connected to the vessel (1), preferably the reservoir (5), wherein fluidic connections between the vessel (1) and the column are provided, in particular wherein the fluidic connections are provided by one or more tubes, optionally further comprising a valve.

[0195] 35. The vessel according to one or more of embodiments 31 to 33, wherein the binding unit (101) is provided as a floating unit configured for float within the reservoir volume (6) and / or the main volume (7).

[0196] 36. The vessel according to one or more of embodiments 31 to 35, wherein the binding unit (101) is by an external loop fluidically connected to the vessel (1) and / or a waste port, preferably both.

[0197] 37. The vessel according to embodiment 36, wherein the external loop comprises at least one valve, preferably one valve for control ling the fluid flow between the binding unit (101) and the waste port and another valve controlled the fluid flow between the vessel (1) and the waste port, preferably wherein both valves are for controlling the fluid flow between the binding unit (101) and the vessel (1) via the external loop.

[0198] 38. A system for cultivating cells in a liquid medium in a cultivation procedure, wherein the system (2) comprises a vessel (1) according to one of embodiments 1 to 37.

[0199] 39. The system according to embodiment 38, wherein the system (2) further comprises: i. a rocker platform (10), preferably wherein the vessel (1) is attached to the rocker platform (10), more preferably such that the rocker platform (10) can pivot the vessel (i); ii. a sensor unit (17), preferably wherein the sensor unit ( 17) is connected to the vessel (1) via the sensor unit connector (20); and / or iii. a heating arrangement, preferably wherein the vessel (1) is heated by the heating arrangement, in particular such that the surrounding area of the vessel (1) is heated. The system according to embodiments 38 or 39, wherein the sensor unit (17) is connected to the vessel (1) in an assembly movement of the vessel (1), in which the vessel (1) is attached to the rocker platform (10). The system according to one or more of embodiments 38 to 40, wherein the heating arrangement is configured for heating a casing part, preferably a lowercasing part (37) and an upper casing part (36), and / or the reservoir (5). The system according to one or more of embodiments 38 to 41, wherein the system further comprises: i. at least one exchangeable bioreactor module comprising a cell vessel area, wherein in the cell vessel area a cell vessel can be located, wherein the cell vessel is a vessel according to one or more of claims 1 to 37 and a media supply area, wherein in the media supply area a media container can be located; ii. a framework, and iii. a transport mechanism for automatically transporting the bioreactor module. The system according to embodiment 42, wherein the transport mechanism is configured to automatically transfer the bioreactor module between different positions, preferably between an expansion location and at least one further position, which can be the refill location, the emptying location, and / or the swapping location. The system according to embodiment 42 or 43, wherein the transport mechanism comprises a transport element that is adapted to hold a bioreactor module and movable in at least one direction, preferably at least one linear direction, such as vertically. The system according one or more of embodiments 42 to 44, wherein the transport mechanism further comprises a further transport element for pushing the bioreactor module into the expansion location and / or for retrieving the bioreactor module from the expansion location. The system according to one or more of embodiments 42 to 45, wherein the bioreactor module further comprises a support structure, wherein due to the support structure the bioreactor module can be handled as a unit, in particular due to the support structure the bioreactor module can be automatically transported by the transport mechanism. 47. The system according to embodiment 46, wherein due to the support structure the bioreactor module can be handled as a unit together with the vessel, the media container and, optionally, a waste container.

[0200] 48. The system according to embodiment 46 or 47, wherein the support structure of the bioreactor module comprises a life support area in which a life support container, in particular a life support tray, can be placed.

[0201] 49. The system according to one or more of embodiments 38 to 48, wherein the system further comprises

[0202] (I) a vessel located in the cell vessel area, a media container located in the media supply area, and optionally a waste container located in a waste area; or

[0203] (II) a vessel located in the cell vessel area, a life support container located in a life support area.

[0204] 50. The system according to embodiment 48 or 49, wherein the life support area comprises the media supply area and / or the waste area, preferably wherein the media supply area is the same area as or a different area than the waste area on the life support area, more preferably wherein the media supply area is the same area as the waste area.

[0205] 51. The system according to one or more of embodiments 48 to 50, wherein the life support area comprises the media supply area and / or the waste area such that the life support container contains a media container and / or a waste container.

[0206] 52. The system according to one or more of embodiments 46 to 51, wherein the support structure further comprises a rocking unit for rocking the vessel.

[0207] 53. The system according to one or more of embodiments 46 to 52, wherein the support structure further comprises a balance for weighing a life support tray.

[0208] 54. The system according to embodiment 53, wherein the balance weighs the life support tray: before a transfer of a liquid medium from the media supply area to the vessel, after a transfer of a liquid medium from the media supply area to the vessel, during a transfer of a liquid medium from the media supply area to the vessel, before a transfer of a liquid from the vessel to the waste area, after a transfer of a liquid from the vessel to the waste area, and / or during a transfer of a liquid from the vessel to the waste area, to determine and / or control an amount of transferred liquid medium. The system according to one or more of embodiments 42 to 54, wherein the system is configured for allowing swapping during a cell culture cycle, in particular automatically in a swapping routine by the system. The system according to one or more of embodiments 42 to 55, wherein the system is configured for allowing swapping of: the media container for a second media container, the waste container for a second waste container, and / or the life support container containing the media container and / or the waste container for a second life support container containing the second media container and / or a second waste container, preferably, wherein swapping is performed manually or automatically, more preferably wherein swapping is performed manually. The system according to one or more of embodiments 42 to 56, wherein the system is configured for performing one or both of the following operations in an automated manner: a refill routine, preferably wherein the system automatically connects a refill container comprising a liquid medium to the media container and automatically refills the media container with the liquid medium and preferably automatically disconnects the refill container from the media container, optionally during a cell culture cycle; and an emptying routine, preferably wherein the system automatically connects a further waste container to the waste container and automatically empties the waste container from the waste and preferably automatically disconnects the further waste container from the waste container, optionally during a cell culture cycle. The system according to one or more of embodiments 42 to 57, wherein the system, preferably the framework of the system, comprises a refill location, at which a refill routine can be performed; an emptying location, at which an emptying routine can be performed; and / or a swapping location, at which a swapping operation can be performed, preferably wherein the system performs the swapping and / or at the swapping location the swapping is performed manually; optionally, wherein the system preferably the framework of the system, comprises the refill location and the emptying location, both of which have the same location. The system according to one or more of embodiments 42 to 58, wherein the system, preferably the framework of the system, comprises at least one expansion location, preferably several expansion locations. 60. The system according to embodiment 59, wherein the framework of the system comprises several expansion locations, wherein the expansion locations are placed vertically, more preferably, the expansion locations are compartments in a shelf with one or more vertical columns.

[0209] 61. The system according to one or more of embodiments 42 to 60, wherein the comprises expanding the cells orthe cell culture, preferably wherein expanding the cells is performed predominantly at an expansion location of the system.

[0210] 62. The system according to one or more of embodiments 59 to 61, wherein the cell expansion location(s) is / are outside a swapping location, an emptying location and / or a swapping location.

[0211] 63. The system according to one or more of embodiments 42 to 62, wherein the system, preferably the framework of the system, comprises a connection arrangement configured for aseptically connecting and / or disconnecting one or more, preferably all, of the following the media container and the vessel, the media container and the refill container, and the waste container and the vessel, preferably, the connection arrangement comprises a tube welding unit for welding together and / or cutting and closing off one or more tubes connected to the media container and the vessel, and preferably the waste container and the vessel.

[0212] 64. The system according to embodiment 63, wherein the connection arrangement is movable, preferably movable in an automated manner, wherein the connection arrangement can be moved to be located at the swapping location, at the refill location, and / or at the emptying location.

[0213] 65. The system according to one or more of embodiments 42 to 64, wherein the bioreactor module, in particular the life support tray, comprises at least one tube holder configured for holding at least one tube, preferably for holding at least one tube in a defined position relative to the support structure and / or the life support container.

[0214] 66. The system according to embodiment 65, wherein the tube holder comprises a tube mover mechanism for extending and / or retracting the at least one tube relative to the tube holder.

[0215] 67. The system according to embodiment 65 or 66, wherein a connection arrangement, preferably a tube welding unit, interacts with the tube holder to weld and / or cut and seal the at least one tube.

[0216] 68. The system according to one or more of embodiments 42 to 67, wherein the bioreactor module comprises a receiving interface with a connection for receiving electrical energy; a connection for receiving wire-bound signals; a connection for receiving gas; and / or a connection for a sensor, such as an optical sensor.

[0217] 69. The system according to one to more of embodiments 42 to 68, wherein the framework comprises providing interfaces providing electrical energy; wire-bound signals; gas; a sensor connection, such as an optical sensor connection, preferably at different locations, in particular at the swapping location, at the expansion location, at the refill location, and / or at the emptying location.

[0218] 70. The system according to embodiment 68 or 69, wherein the receiving interface is connectable to the providing interface of the framework.

[0219] 71. The system according to one or more of embodiments 42 to 70, wherein the bioreactor module further comprises a lid for providing a controlled environment for the vessel; and / or a heating unit for the vessel.

[0220] 72. The system according to one or more of embodiments 42 to 71, wherein the framework comprises a unit operation location, in particular next to a swapping location or a refill location, preferably wherein at the unit operation location the cell culture is transferred from a cell container of a cartridge into the vessel.

[0221] 73. A method for cultivating cells in a liquid medium comprising: a) providing cells in a liquid medium; b) cultivating the cells using a vessel (1) according to one or more of embodiments 1 to 37 and / or a system according to one or more of embodiments 38 to 72.

[0222] 74. The method according to embodiment 73, wherein the cells are dedicated for cell and / or gene therapy, preferably dedicated for cell therapy.

[0223] 75. The method according to embodiment 73 or 74, wherein the cells are immune cells, preferably T cells.

[0224] 76. The method according to one or more of embodiments 73 to 75, wherein the cells are genetically modified T cells, preferably genetically modified CAR-T cells.

[0225] 77. The method according to one or more of embodiments 73 to 76, wherein the cells become genetically modified, preferably, wherein the method comprises providing an agent for genetically modifying the cells present in the vessel (1), more preferably wherein the cells are T cells. The method according to one or more of embodiments 73 to 77 , wherein step (b) comprises

[0226] (b.l) a preliminary cultivation phase, in which the cells are preliminarily cultivated, preferably wherein cells are predominantly cultivated in the reservoir (5); and

[0227] (b.2) a subsequent cultivation phase, in which the cells are subsequently cultivated preferably wherein cells are cultivated in the reservoir (5) and the casing (4). The method according to one or more of embodiments 73 to 78, wherein the reservoir (5) defines a reservoir volume (6) for receiving the liquid medium in the preliminary cultivation phase, wherein the casing (4) defines a main volume (7) for receiving the liquid medium in the subsequent cultivation phase. The method according to one or more of embodiments 73 to 79, wherein the method further comprises one or more of the following steps: optionally, selecting cells one or more times, in particular by binding at least a fraction of the cells expressing a ligand, preferably selecting a subpopulation of T cells; activating cells, preferably activating T cells; transducing cells, preferably transducing T cells; transfecting cells, preferably transfecting T cells; and / or expanding cells, preferably T cells, more preferably genetically modified T cells; preferably, wherein the method comprises at least two, three or four of the above- mentioned steps. The method according to one or more of embodiments 73 to 80, wherein the method comprises after step a) but before step b): optionally, selecting cells one or more times, preferably by binding at least a fraction of the cells expressing a ligand; activating cells, preferably activating T cells; and transducing cells, preferably transducing T cells, or transfecting cells, preferably transfecting T cells. A method for manufacturing a cell therapy product comprising: a) providing cells in a liquid medium; b) cultivating the cells using a vessel (1) according to one or more of embodiments 1 to 37 and / or a system according to one or more of embodiments 38 to 72. The method according to embodiment 82, wherein the cells are immune cells, preferably T cells. The method according to embodiment 82 or 83 wherein the cells are genetically modified T cells, preferably genetically modified CAR-T cells. -

[0228] 85. The method according to one or more of embodiments 82 to 84, wherein the method further comprises one or more of the following steps: optionally, selecting cells one or more times, in particular by binding at least a fraction of the cells expressing a ligand, preferably selecting a subpopulation of T cells; activating cells, preferably activating T cells; transducing cells, preferably transducing T cells; transfecting cells, preferably transfecting T cells; and / or expanding cells, preferably T cells, more preferably genetically modified T cells; preferably, wherein the method comprises at least two, three or four of the above- mentioned steps.

[0229] 86. The method according to one or more of embodiments 82 to 85, wherein the method comprises after step a) but before step b): optionally, selecting cells one or more times, preferably by binding at least a fraction of the cells expressing a ligand; activating cells, preferably activating T cells; and transducing cells, preferably transducing T cells, or transfecting cells, preferably transfecting T cells.

[0230] 87. The method according to one or more of embodiments 82 to 86, wherein the method further comprises after step b) formulating the expanded cell culture into a media suitable for being injected into a patient and / or transferring the expanded cell culture from the vessel into a harvesting vessel.

[0231] 88. Use of the vessel according to one of embodiments 1 to 37 and / or system according to one or more of embodiments 38 to 72 for manufacturing a cell therapy product, preferably wherein manufacturing comprises at least one of the following: optionally, selecting cells one or more times, in particular by binding at least a fraction of the cells expressing a ligand, preferably selecting a subpopulation of T cells; activating cells, preferably activating T cells; transducing cells, preferably transducing T cells; transfecting cells, preferably transfecting T cells; and / or expanding cells, preferably T cells, more preferably genetically modified T cells.

[0232] 89. Use of the vessel according to one of embodiments 1 to 37 and / or system according to one or more of embodiments 38 to 72 in a method according to one or more of embodiments 73 to 87.

[0233] 90. Cultivated cells obtained by the method according to one or more of embodiments 73 to 81. 91. A cell therapy product obtained by the method according to one or more of embodiments

[0234] 82 to 87.

[0235] Further aspects and features of the present disclosure

[0236] The main realization of the present invention is that the reservoir is pre-shaped, making it easy to handle and dimensionally stable. This achieves a high reproducibility for a critical processes like cell and gene therapy manufacturing. Whereas reservoirs of vessels known in the art are shaped temporarily by a rocking motion platform or stage, the reservoir of the provided vessel is permanently shaped. The shape of the reservoir might be optimized according to the cultivation procedure and / or the used cells and / or the used liquid medium. Because of the pre-shaped reservoir, which may correlate with a pre-shaped reservoir volume, several advantages regarding evaporation, nutrient supply and / or measurement occur. It also becomes easier to drain the vessel fully, for example via ports attached to the reservoir. For example, during the cultivation procedure the volume of the liquid medium in the vessel increases over time. In the preliminary cultivation phase the volume of the liquid medium is rather small compared to the volume in the subsequent cultivation phase. Here, the liquid medium containing the cells can be held mainly, preferably completely, in the reservoir. As a result, evaporation might be minimized or at least reduced due to an optimized surface to volume ratio of the liquid medium. However, it is possible to further control evaporation and condensation, for example by using a condensate arrangement. Furthermore, the nutrient supply might be improved since mixing of the liquid medium in the reservoir is improved. If needed, also measuring properties of the cells and / or the liquid medium can be improved. Since the liquid medium can be held mainly in the reservoir in a preliminary cultivation phase, accurate measurement might be possible even with small volumes of liquid medium containing the cells. A preferred reservoir-shape also ensures liquid contact with only a small surface area of reservoir material. This ensures reduced movement of any leachables or extractables from the reservoir material, because this movement is driven by surface area in contact.

[0237] In detail, it is proposed that the reservoir is a pre-shaped reservoir.

[0238] A further embodiment relates to a harvesting connection arrangement. The cells may be drained through a harvesting port, preferably from a local minimum to improve the harvesting, for example such that a maximum of liquid medium containing the cells can be drained from the vessel. For supplying liquid medium and / or chemicals, etc. to the vessel, a supply connection arrangement for fluidically connecting a supply is preferred. To guarantee good supply and mixing, the liquid medium and / or cells and / or chemicals may be introduced directly into the reservoir volume via a supply port at a supply location.

[0239] In a further embodiment, the casing comprises an upper casing part and a lower casing part and the reservoir may be attached to the lower casing part in different ways.

[0240] In a further embodiment, the vessel comprises a main membrane which may be used for perfusion in particular.

[0241] In a further preferred embodiment, the vessel is pre-sterilized and intended for single-use application. This may lead to more reliable cultivation procedures and avoidance of contamination when using the vessel.

[0242] A further embodiment relates to a reservoir membrane which may allow cultivating the cells in the reservoir volume already via perfusion, but also introducing media through the membrane, media exchanges or the like.

[0243] A further embodiment describes preferred geometric embodiments. Such embodiment refers to a preferred embodiment with a sensor element and the location of the sensor element.

[0244] A further embodiment relates to the reservoir being transparent for a sensor, which simplifies the production of the reservoir.

[0245] Another embodiment relates to a tube loop leading from and to the vessel. The loop may be used for cell counting, for example.

[0246] Several aspects of the proposed vessel are relevant independently of the pre-shaped reservoir, preferably but not necessarily in combination with compartments like the reservoir and the casing for the reservoir volume and the main volume. Such aspects are for example the existence of a membrane in the reservoir, the existence of two membranes, the loop for imaging or the use of the vessel for cell and gene therapy. Several advantageous aspects are described with regard to cell and gene therapy in particular, which are of importance. For example, a vessel for cultivating cells in a liquid medium in a cultivation procedure, wherein the cultivation procedure comprises a preliminary cultivation phase, in which the cells are preliminarily cultivated, and a subsequent cultivation phase, in which the cells are subsequently cultivated, wherein the vessel comprises two distinct compartments, one with a reservoir volume for receiving the liquid medium in the preliminary cultivation phase and one with a main volume for receiving the liquid medium in the subsequent cultivation phase, wherein the reservoir volume and the main volume are adjacently arranged and are fluidically connected for fluidical exchange, is relevant in combination with several of the features described herein. In particular, it may be the case that the vessel comprises a main membrane contacting the liquid medium in the main volume and a reservoir membrane contacting the liquid in the reservoir volume. The membranes may both be used for perfusion or may be used for different functionalities.

[0247] Further relevant is a vessel which comprises a tube loop leading from the vessel and to the vessel, with or without two distinct volumes. In combination with for example the rocker table, the cell and gene therapy use case, the branches, etc., these features also have relevance on their own.

[0248] The same is also true for vessel adapted to be used or a method for using a vessel in which air drawn from the vessel is used to remove liquid residues from one or more tubes attached to the vessel.

[0249] Another teaching, which is of equal importance, relates to a system for performing a cultivation procedure for cultivating cells in a liquid medium, wherein the system comprises a vessel as proposed. The system might be a rocking motion bioreactor.

[0250] All explanations given with regard to the proposed vessel are fully applicable to such system.

[0251] A further embodiment relates to improved embodiments of the system.

[0252] Another teaching, which is of equal importance, relates to a method for cultivating cells in a liquid medium using a vessel as proposed.

[0253] All explanations given with regard to the proposed vessel and the proposed system are fully applicable to such method.

[0254] In a further embodiment, the cells are cultivated for cell and / or gene therapy. Novel approaches for cell and gene therapy pose several challenges that need to be solved with the right combination of automated or semi-automated systems, bioreactors and othervessels, cultivation conditions, flexibility and the like.

[0255] Description of Figures

[0256] Disclosed herein is a vessel 1 for cultivating cells in a liquid medium in a cultivation procedure. Preferably, the vessel 1 is a bag, which is flexible. Flexible does not necessarily mean collapsible. In Fig. 1 the vessel 1 is used in a system 2 for performing the cultivation procedure for cultivating the cells in the liquid medium. The system 2 may be an integrated bioprocessing system for cells destined forcell and / or gene therapy, in particular immune or naive cells. The system 2 preferably comprises a tray 3 for cultivating the cells by imposing a rocking motion onto the vessel 1 during the cultivation procedure. The term "rocking motion" includes all types of rocking motion, whether strictly rocking or more wave-like. In figures 2a), 2b), 3a), 3b), 3c) the vessel 1 is shown in more detail.

[0257] The cultivation procedure may comprise a preliminary cultivation phase and a subsequent cultivation phase. During the preliminary cultivation phase the cells are preliminarily cultivated, for example such that a certain amount or concentration of cells in a certain volume of liquid medium may be achieved. During the subsequent cultivation phase the cells are subsequently cultivated, in particular subsequent to a preliminary cultivation phase, wherein, for example, the volume of liquid medium is increased substantially to reach higher amounts of cells and / or to keep the cells viable for an extended period of time. Generally, the volume of liquid medium containing the cells increases from the preliminary cultivation phase to the subsequent cultivation phase, wherein it is not excluded that the volume increases within each of the phases itself. The terms "preliminary" and "subsequent" describe the order of the phases and not necessarily the cultivation itself. It is preferred though that the cultivation is performed differently in the two phases, for example with different feed strategies.

[0258] The vessel 1 comprises a casing 4 and a reservoir 5, which are depicted for example in a first embodiment in Figs. 2 and 3. Figs. 4 to 6 show another embodiment. All features described for one embodiment may be present in the other embodiment and can be combined in any sensible way.

[0259] The reservoir 5 defines a reservoir volume 6, in which the liquid medium containing the cells can be received in the preliminary cultivation phase and preferably also in the subsequent cultivation phase. The casing 4 defines a main volume 7 in which the liquid medium can be received in the subsequent cultivation phase. Gas might be introduced to the main volume 7, too. For this, the vessel 1 may comprise a gas inlet port 8. Further, gas may also be removed from the vessel 1. For this, the vessel 1 may comprise a gas outlet port 9. The casing 4 may be, like here, flexible or alternatively rigid.

[0260] The reservoir volume 6 may hold a maximum volume of liquid medium of at most 250 ml, preferably 200 ml, more preferably 150 ml, more preferably 120 ml, e.g., 100 ml or 80 ml. As it can be seen exemplarily in Figs. 2a) and 3b), the main volume 7 is preferably larger than the reservoir volume 6, wherein it is preferred that the main volume 7 is at least three times, preferably at least five times, more preferably at least eight times the reservoir volume 6. The main volume 7 may be at most 6 I, preferably 4 I, more preferably 3 I, more preferably 2 I, more preferably 1 1. It is possible that the main volume 7, during the cultivation procedure, is only partly filled with liquid medium. Here, it is possible that the main volume 7 may hold a volume of liquid medium of at most 3 I, preferably 2,5 I, more preferably 2 I, e.g., 1,5 I, 1,25 I, 1 I, or 0,5 I. It is in general preferred that the maximum volume of liquid medium, which is held, is at most half of the main volume 7. The reservoir volume 6 and the main volume 7 both are inner Ovolumes of the vessel 1.

[0261] The reservoir volume 6 and the main volume 7 are typically adjacently arranged and are fluidically connected for fluidical exchange. "Fluidical exchange" in this context may comprise that liquid medium located in the reservoir 5 and liquid medium located in the casing 4 can be mixed such that an exchange between the liquids located in the different volumes happens. In the preliminary cultivation phase, liquid medium might be contained solely in the reservoir 5, whereas fluidical exchange may happen mostly in the subsequent cultivation phase. The reservoir volume 6 and the main volume 7 develop the overall volume of the vessel 1. Also, in the preliminary cultivation phase liquid may enter the main volume 7, in particular during rocking. The transition between the preliminary cultivation phase and the subsequent cultivation phase may be a continuous transition if for example culture medium is added continuously and not in batches.

[0262] Proposed is that the reservoir 5 is a pre-shaped reservoir 5.

[0263] Thus, the reservoir 5 of the vessel 1 is dimensionally stable such that the reservoir volume 6 is fully developed before the vessel 1 is attached to the rocker platform 10 of the system 2. "Pre-shaped" does not exclude that the reservoir 5 can be generally deformed, however means that the reservoir 5 has a certain defined shape. It is possible that the reservoir 5 returns to its pre-shaped form when a deformation force decreases or disappears.

[0264] The reservoir 5 is preferably rigid. Thus, the reservoir 5 has a certain defined shape, which is basically dependent on the production design of the reservoir 5, and cannot be deformed considerably. "Rigid" may not exclude that the reservoir 5 is elastic to a certain degree at all, but means that the pre-shaped form of the reservoir 5 basically cannot be non-destructively changed, e.g. collapsed.

[0265] The reservoir 5 is attached to the casing 4, preferably such that the reservoir volume 6 extends the main volume 7, wherein, more preferably, the reservoir volume 6 and the main volume 7 develop the overall volume of the vessel 1.

[0266] Generally, different designs of the reservoir 5 are possible. For example, the reservoir 5 may comprise a single section, which can evenly be filled with the liquid medium containing the cells. When the reservoir volume 6 is completely filled, further introduction of liquid medium leads to overflowing of liquid medium to the main volume 7 (as shown in the second embodiment). However, regarding the design of the reservoir 5, it is preferred in the first embodiment that the reservoir 5 comprises two sub-sections 11, wherein each sub-section 11 defines a sub-volume 12 of the reservoir volume 6 and wherein the two sub-volumes 12 are fl uid ically connected, as it can be exemplarily seen in figures 3a), 3b) and 3c). The sub-volumes 12 are part of the reservoir volume 6. The sub-volumes 12 are f I uidically connected such that a fluidical exchange among the sub-sections 11 is possible. In particular, the sub-sections 11 are designed as structurally separate sub-sections 11, as it can be seen in fig. 3a).

[0267] In a preferred embodiment, one of the sub-sections 11 is a sensor section 13 defining a sensor volume 14. Here, the sensor volume 14 is one of the sub-volumes 12. The sensor section 13 and the sensor volume 14 might be designed such that measurements of the cells and / or the liquid medium in the sensor volume 14 can be made. In view of a sensor arrangement 15 of the vessel 1, which will be described later on, it is particularly advantageous that the sensor section 13 provides a constant sensor volume 14 and thus a constant sensor field of view to a sensor that does not change when the filling volume of the liquid medium is increased. The sensor might be formed by a sensor element 16 or sensor elements 16 of the sensor arrangement 15 and a sensor unit 17, which is attached to the vessel 1. The sensor might be a capacitance-based sensor. The reservoir 5 and particularly the sensor section 13 can provide the required sensor volume 14 to the capacitance-based sensor at a much lower fill volume of the liquid medium compared to a "traditional" vessel 1 without a pre-shaped reservoir 5.

[0268] Alternatively or additionally, it is preferred that one of the sub-sections 11 is a funnel section 18 defining a funnel volume 19. The funnel volume 19 is one of the sub-volumes 12. The funnel section 18 with regard to the other one of the sub-sections 11 is preferably designed and arranged such that the liquid medium containing the cells can gravitate from the funnel section 18 to the other one of the sub-sections 11. "Gravitate" in this context means that the liquid medium from the funnel section 18 tends to flow in the direction of the other one of the subsections 11 and particularly into the other one of the sub-sections 11 due to the gravitational force, if the vessel 1 is oriented horizontally. This effect might be enhanced due to the rocking motion.

[0269] Here and preferably, one of the sub-sections 11 is the funnel section 18 and the other one of the sub-sections 11 is the sensor section 13, as it can be exemplarily seen in Figs. 3b) and 3c). Here and preferably, the sensor section 13 is stepped towards the funnel section 18 in the vertical direction such that the lowest level of the sensor section 13 is below the lowest level of the funnel section 18 with regard to the horizontal plane. Here and preferably, the sensor volume 14 is smaller than the funnel volume 19, in particular such that also small amounts of the liquid medium containing cells may be accurately measured in the sensor section 13.

[0270] It should be noted that for the main volume 7 and the reservoir volume 6 and also for the subsections 11, these cannot be defined arbitrarily. The vessel 1 may have some sort of visible and / or functional boundary between the sections and volumes.

[0271] As mentioned beforehand in another context, in particular with regard to the sensor section 13, it is preferred that the reservoir 5 comprises a sensor arrangement 15. The sensor arrangement 15 is preferably attached to the sensor section 13, as it is the case for the preferred embodiment (Fig. 3c)).

[0272] Here and preferably, the sensor arrangement 15 comprises a sensor unit connector 20 (Fig. 2b)) for connecting a sensor unit 17, in particular to the reservoir 5, preferably to the sensor section 13, such that properties of the cells and / or the liquid medium can be measured. The sensor unit 17 may be an external sensor unit 17, for example of the system 2. It is possible that the sensor unit 17 comprises a sensor, in particular a capacitance-based sensor, like a biomass sensor for measurement of the biomass concentration, particularly the viable cell concentration in the liquid medium in the vessel 1. The sensor unit connector 20 may be designed as a plug-in connector. It is generally possible that the sensor unit connector 20 comprises spring-loaded pins. The sensor unit connector 20 is preferably attached to the reservoir 5, as it can be seen in Fig. 2b). The sensor unit connector 20 is here and preferably attached to the sensor section 13.

[0273] Alternatively or additionally to the sensor unit connector 20, the sensor arrangement 15 comprises one or several sensor elements 16 (Fig. 3b)) for measuring properties of the cells and / or the liquid medium. Here, the sensor arrangement 15 comprises four sensor elements 16. The sensor element 16 or the sensor elements 16 might be electrodes, like it is the case for the preferred embodiment. In this case, it is possible that the sensor unit 17 and the sensor element 16 or the sensor elements 16 are part of a sensor, like a biomass sensor. Here and preferably, the sensor unit 17 and the sensor elements 16 are part of a capacitance-based sensor for measurement of the biomass, in particular a concentration of viable cells. Such a sensor might be a Viamass sensor, for example, as made by Aber Instruments.

[0274] The sensor element 16 or the sensor elements 16 are preferably attached to the sensor section 13, particularly to a side wall of the sensor section 13 (Fig. 3b)) or to a bottom wall of the sensor section 13. Attaching the sensor element 16 or sensor elements 16 to the side wall of the sensor section 13 can improve measurement accuracy, since deposition of cells might be minimized or avoided. Attaching the sensor element 16 or sensor elements 16 to the bottom wall of the sensor section 13 can simplify connection to the sensor unit 17 via the sensor unit connector 20. The sensor element 16 or the sensor elements 16 might be molded into the reservoir 5, particularly the sensor section 13.

[0275] The sub-volumes 12 are fluidically connected. In this regard, it is proposed, that the reservoir 5 comprises a channel section 21, wherein the channel section 21 fluidically connects the subsections 11, in particular the sensor section 13 and the funnel section 18. The channel section 21 can be best seen in Fig. 3a). Preferably, the channel section 21 fluidically connects the subsections 11 such that liquid medium containing cells can flow from one of the sub-sections 11, in particular the funnel section 18, to the other of the sub-sections 11, in particular the sensor section 13. Thus, fluidical exchange of the liquid located in the sub-volumes 12 can happen. As it can be seen in Fig. 3a) and preferably, the channel section 21 comprises a narrowing 22, for example for local acceleration of the flow of liquid medium and / or for minimizing or reducing cell settling and / or for increasing the volume of liquid medium being held in one of the sub-sections 11, in particular in the sensor section 13. Due to the acceleration of the liquid medium in the channel section 21, the local flow velocity may rise. "Local" in this context means that the acceleration and thus the higher velocity of the liquid medium is limited to a part of the channel section 21 nearby the narrowing 2 . Generally, the sub-sections 11 and the channel section 21 preferably are designed integrally. The sub-sections 11 and the channel section 21 preferably develop a basin-like reservoir 5.

[0276] According to one embodiment, the reservoir 5 comprises a longitudinal extension 23 and the sub-sections 11, in particular the sensor section 13 and the funnel section 18, are arranged next to each other with regard to the longitudinal extension 23 such that a main flow direction of liquid medium containing cells within the reservoir 5 is defined. The longitudinal extension 23 is the maximum extension of the reservoir volume 6 in the longitudinal direction. The reservoir 5 may comprise a transversal extension 24, which is orientated transversally in comparison to the longitudinal extension 23, but still in the same horizontal plane. The transversal extension 24 is the maximum extension of the reservoir volume 6 in the transversal direction. The longitudinal extension 23 is, as it can be seen for example in Fig. 3a), larger than the transversal extension 24, preferably the longitudinal extension 23 is at least two times, more preferably at least four times, more preferably at least six times, the transversal extension 24.

[0277] In this context it is furthermore preferred that the sensor arrangement 15, in particular the sensor unit connector 20 and / or the sensor element 16 or the sensor elements 16, is or are attached to the sensor section 13 transversely to the longitudinal extension 23, preferably in the horizontal direction, and, in particular, transversely to the main flow direction.

[0278] As the narrowing 22 of the channel section 21, as described beforehand, can already lead to better mixing of the liquid medium in the reservoir volume 6, it is independently thereof preferred that the reservoir 5 comprises a baffle 25. The baffle 25 extends at least partly into the reservoir volume 6, preferably into the channel section 21, more preferably into the narrowing 22, such that a bottom flow of liquid medium containing cells can be developed within the reservoir volume 6, in particular within one of the sub-sections 11, in particular within the sensor volume 14. This way, cell settling may be minimized or at least reduced. As it can be seen in Figs. 3b) and 3c) and preferably, the baffle 25 extends into the narrowing 22 of the channel section 21 and locally reduces the flow area through the channel section 21. An upper part of the narrowing 22 is mostly covered by the baffle 25, wherein a lower part of the narrowing 22 is not covered by the baffle 25. The baffle 25 might be designed plate-like, wherein it particularly extends transversely with regard to the main flow direction and / or the longitudinal extension 23 of the reservoir 5. It is possible that the baffle 25 decelerates the overall volume flow from the sensor section 13 to the funnel section 18 in the preliminary cultivation phase during the rocking motion. This might improve measurements by a sensor unit 17 attached to the sensor unit connector 20 and / or located in the sensor section 13.

[0279] Regarding the baffle 25, it proved beneficial if the baffle 25 extends at least partly into the main volume 7, particularly such that a circuit flow of liquid medium containing cells can be developed within the reservoir volume 6 and the main volume 7 in the subsequent cultivation phase. This is also the case for the preferred embodiment in the figures, as it can be seen in Figs. 3a), 3b) and 3c).

[0280] As described already beforehand, the reservoir 5 may comprise a sensor arrangement 15 with a sensor unit connector 20 for connecting a sensor unit 17 and / or with a sensor element 16 or sensor elements 16 for measuring properties of the cells and / or liquid medium, particularly the viable cell concentration in the liquid medium. However, it is possible and preferred that the reservoir 5 comprises a further sensor arrangement 26. The further sensor arrangement 26 is preferably attached to the channel section 21 (Fig. 3b)) or to one of the sub-sections 11, particularly the sensor section 13. The further sensor arrangement 26 can be arranged such that the properties of the liquid medium containing the cells, which passes the channel section 21, particularly the narrowing 22, can be measured.

[0281] It is especially preferred that the sensor arrangement 15 and the further sensor arrangement 26 build a sensor assembly group. This way, the sensor arrangement 15 and the further sensor arrangement 26 can be manufactured and tested as a structural unit before being connected to the reservoir 5. Designing the sensor arrangements 15, 26 such that the sensor assembly group is build has furthermore the advantage that sealing is simplified. Particularly, only one seal is required when connecting the sensor assembly group to the reservoir 5. If the sensor arrangements 15, 26 were left separately, separate seals, potentially increasing the likelihood of leaks, would be required.

[0282] Preferably, the further sensor arrangement 26 comprises a further sensor unit connector 27 for connecting a further sensor unit 28, in particular to the reservoir 5, such that properties of the cells and / orthe liquid medium can be measured. The further sensor unit 28 might be an external further sensor unit 28, for example of the system 2. It is possible that the further sensor unit 28 comprises a further sensor, in particular an optical-sensor, like a pH-sensor, a dissolved oxygensensor, or the like. The optical-sensor can in particular be designed as an optode. The further sensor unit connector 27 may be designed as a plug-in connector. It is generally possible that the furthersensor unit connector 27 comprises spring-loaded pins. The further sensor unit connector 27 is preferably attached to the reservoir 5 as can be seen in Fig. 2b). The further sensor unit connector 27 is here and preferably attached to the channel section 21, adjacent to the sensor section 13. The further sensor unit connector 27 might be attached to one of the sub-sections 11, in particular to the sensor section 13.

[0283] Alternatively or additionally to the further sensor unit connector 27, the further sensor arrangement 26 comprises at least one further sensor element 29 (Fig. 3b)) for measuring properties of the cells and / or the liquid medium. In the embodiment according to Fig. 3b), the further sensor arrangement 26 comprises two further sensor elements 29. Here, the further sensor arrangement 26 comprises a further sensor element 29, which enables pH- and / or dissolved oxygen-measurements. The further sensor element 29 comprises a sensing foil and / or an optical window. The sensing foil faces the reservoir volume 6 such that it may be contacted by the liquid medium containing the cells. It is also possible, that the further sensor arrangement 26 comprises a further sensor element 29 or further sensor elements 29, for example a temperature sensor for measurement of the temperature, etc. The sensor arrangement 15 and / or the further sensor arrangement 26 may generally comprise further components, like a thermowell.

[0284] In this case, wherein the further sensor arrangement 26 comprises at least one further sensor element 29, it is possible that the further sensor unit 28 and the further sensor element 29 develop a further sensor, particularly a pH- or dissolved oxygen-sensor. Preferably, the further sensor unit 28 and the further sensor element 29 develop an optical sensor, particularly an optode. Such a further sensor might be an optode for measuring the pH or dissolved oxygen concentration of the liquid medium in the reservoir volume 6. The furthersensor arrangement 26 may be present without the sensor arrangement 15 being present.

[0285] The further sensor element 29 is attached to the channel section 21 or to one of the sub-sections 11, particularly to the sensor section 13. It is preferred that the further sensor element 29 is attached to a side wall of the channel section 21 (as it can be seen in Fig. 3b)) or the one of the sub-sections 11. Alternatively, the further sensor element 29 can be attached to a bottom wall of the channel section 21 or to one of the sub-sections 11. The further sensor element 29 might be molded into the reservoir 5, particularly the channel section 21 or the sensor section 13.

[0286] Also in this context it is possible that the further sensor arrangement 26, in particularthe further sensor unit connector 27 and / or the further sensor element 29, is or are attached to the channel section 21 or one of the sub-sections 11, particularly the sensor section 13, transversely to the longitudinal extension 23, preferably in the horizontal direction, and, in particular, transversely to the main flow direction.

[0287] At a certain point during the cultivation procedure and / or after the cultivation procedure was performed, harvesting the liquid medium containing the cells is required. For this reason, it is proposed that the reservoir 5 comprises a harvesting connection arrangement 30 for draining the cells from the reservoir volume 6. In this context, it is preferred that the reservoir volume 6 comprises a harvesting location 31 and that the harvesting connection arrangement 30 comprises a harvesting port 32 for draining the cells in the liquid medium at the harvesting location 31, wherein the harvesting port 32 is fluidically connected to the reservoir volume 6, in particular to one of the sub-volumes 12, here and preferably to the sensor volume 14. As it can be seen in Fig. 3c) and preferably, the harvesting port 32 is attached to the sensor section 13, in particular to a side wall of the sensor section 13. It is especially preferred that the harvesting location 31 is a local minimum of the reservoir 5 or reservoir volume 6, more preferably a global minimum, with regard to the horizontal plane. For example, the harvesting location 31 may be the bottom of the sensor volume 14, as it is the case for the preferred embodiment and can be seen in Fig. 3c). The minimum may become a minimum by setting the rocker platform 10 to a certain angle.

[0288] It is possible that the harvesting connection arrangement 30, in particularthe harvesting port 32, is attached transversely to the longitudinal extension 23 and, in particular, to the main flow direction. It is preferred that the harvesting connection arrangement 30 is attached such that the draining direction, which is the direction in which the cells and / or liquid medium is drained out of the reservoir volume 6, in particular the section volume, is essentially orientated horizontally. Alternatively, the harvesting connection arrangement 30, in particularthe harvesting port 32, can be attached such that the draining direction is essentially orientated vertically. "Essentially orientated horizontally" or "essentially orientated vertically" in this context means that the harvesting direction deviates from the horizontal direction or the vertical direction respectively by at most 5°. However, the draining direction can also be at any angle between a horizontal direction and a vertical direction. The harvesting connection arrangement 30, in particular the harvesting port 32, may also be used to draw a sample of cells and / or liquid medium from the bag during the cultivation procedure.

[0289] Besides harvesting, supply of liquid medium and / or cells and / or chemicals impacts or impact the cultivation procedure. For that reason, it is proposed that the reservoir 5 comprises a supply connection arrangement 33 for fluidically connecting a supply to the reservoir volume 6. The fluidic connection may for example be realized by a flexible tube or the like. It is preferred that the reservoir volume 6 comprises a supply location 34 and that the supply connection arrangement 33 comprises at least one, here and preferably two, supply port 35 for supplying liquid medium and / or cells and / or chemicals, etc. at the supply location 34.

[0290] The supply port 35 is fluidically connected to the reservoir volume 6, in particular to the volume of the channel section 21. Thus, cells and / or liquid medium and / or chemicals can be injected directly to the reservoir volume 6 or particularly to the volume of the channel section 21. As it can be seen in Fig. 3c) and preferably, the supply port 35 is or rather supply ports 35 are attached in the channel section 21, in particular to a side wall of the channel section 21. It is especially preferred that the supply location 34 is a location in the channel section 21.

[0291] It is possible that the supply connection arrangement 33, in particular the supply port 35, is attached transversely to the longitudinal extension 23 and, in particular, to the main flow direction. It is preferred that the supply connection arrangement 33 is attached such that the supply direction, which is the direction in which the cells and / or liquid medium and / or the chemicals is or are injected to the reservoir volume 6, particularly to the volume of the channel section 21, is essentially orientated horizontally. Alternatively, the supply connection arrangement 33, in particular the supply port 35, can be attached such that the supply direction is essentially orientated vertically. As beforehand, "essentially orientated horizontally" or "essentially orientated vertically" in this context means that the supply direction deviates from the horizontal direction or rather the vertical direction at most 5°. However, the supply direction can also be at any angle between a horizontal direction and a vertical direction.

[0292] It may also be the case, that one of the supply ports 35 is used for supplying liquid medium and / or cells and / or chemicals, etc. at the supply location 34, while the other one of the supply ports 35 is used for sampling a representative sample from the bag.

[0293] While most of the described features relate mainly to the reservoir 5 itself, the design of the casing 4 may improve the vessel 1 further. Regarding the casing 4, it is especially preferred that the casing 4 comprises an upper casing part 36 and a lower casing part 37, wherein the reservoir 5 is attached to the lower casing part 37. "Upper" and "lower" refers to the horizontal plane, as it can be identified, for example, in Figs. 2a), 2b) and 3a), 3b), 3c). With regard to the vertical direction, the upper casing part 36 and the lower casing part 37 are thus arranged adjacently. The upper casing part 36 and the lower casing part 37 might be formed integrally, for example by using a plastic injection molding process or by a thermoforming procedure. The upper casing part 36 and the lower casing part 37 may be designed two-parted, wherein the upper casing part 36 and the lower casing part 37 are connected, in particular welded, for example by using a plastic welding process.

[0294] The reservoir 5 and the lower casing part 37 are connected. The reservoir 5 and the lower casing part 37 may be welded, which might be preferably the case if the lower casing part 37 and the reservoir 5 comprise the same material, for example plastic, particularly polyethylene. Alternatively or additionally, it is also possible that the reservoir 5 comprises an attachment arrangement 38 for attaching the reservoir 5 to the lower casing part 37, like it is seen in Figs. 3a), 3b) and 3c). Here and preferably, the attachment arrangement 38 comprises at least two attachment elements 39, wherein a part of the lower casing part 37 is clamped between the attachment elements 39. It is possible that the attachment arrangement 38 comprises a sealing, like an O-ring or the like. It is also possible that the reservoir 5 or at least a part of the reservoir 5, like the sub-sections 11 and the channel section 21 of the reservoir 5, and the lower casing part 37 are designed integrally. Here and preferably, the reservoir 5 is attached to the lower casing part 37 such that the reservoir volume 6 of the reservoir 5 is arranged below the main volume 7 of the casing 4 with regard to the horizontal plane. Preferably, the reservoir 5 is attached to the lower casing part 37 by welding or formed integrally with the lower casing part 37, in particular by injection molding and / or thermoforming and / or gluing, for example UV cured gluing.

[0295] Generally, the casing 4, particularly the lower casing part 37, may be pre-shaped and rigid, like the reservoir 5. However, it is also possible that the casing 4, particularly the lower casing part 37, is flexible and particularly obtains its form during the cultivation procedure, for example because of rising pressure or weight inside the vessel 1. In both cases, the lower casing part 37 is preferably designed funnel-like such that liquid medium containing cells can gravitate from an inner surface of the lower casing part 37 in the inner main volume 7 to the reservoir volume 6. The funnel-like design of the lower casing part 37 can be best seen in Figs. 3a), 3 b) and 3 c). The funnel-like design of the lower casing part 37 may be present only in a mounted state if the casing 4 is collapsible as is the case in one embodiment. In another embodiment, the casing 4 is not collapsible.

[0296] According to one preferred embodiment, the vessel 1 comprises a main membrane 40. The main membrane 40 may be in direct contact with liquid in the main volume 7. The main membrane 40 may be part of a perfusion arrangement 41 and / or may form a boundary of the main volume 7 and / or may be used for cultivating the cells in the liquid medium via perfusion in the main volume 7 in the subsequent cultivation phase, which is preferably performed as a step in the method disclosed herein. Through the main membrane 40, the cells in the liquid medium may be retained in the main volume 7 during perfusion. The main membrane 40 may be a PES membrane with a cutoff of 1.2 micrometer. It is preferred that the main membrane 40 is arranged at the lower casing part 37 surrounding the reservoir 5 or, as it is the case for the preferred embodiment in the figures, adjacently to the reservoir 5. It is possible that the vessel 1 comprises a waste port (not shown) for draining liquid medium, which was filtered by the main membrane 40 and is therefore cell-free. The membrane may also be a floating membrane.

[0297] According to another embodiment it is proposed that the vessel 1 comprises a condensate arrangement for aimed local condensation of evaporated liquid medium, wherein the condensate arrangement comprises a coolable draining element, which particularly is cooled in the cultivation procedure, preferably at least in a preliminary cultivation phase. Preferably, the draining element is arranged above the reservoir 5 such that condensed liquid medium can drain, in particular drop, into the reservoir volume 6, in particular in the preliminary cultivation phase. The draining element may for example be designed as a cold finger. Additionally or alternatively, the surface of the casing 4 may be heated such that condensation is prevented or at least reduced.

[0298] According to one further embodiment it is proposed that the vessel 1 is single-use and presterilized such that the inner main volume 7 and the reservoir volume 6 are sterile. This is of course the case prior to introducing the cells or anything else. The vessel 1 might be sterilized during production.

[0299] Turning now to the second embodiment in Figs. 4 to 6, it may be the case that the reservoir 5 comprises a reservoir membrane 42 forming a at least a part of the boundary of the reservoir volume 6. The reservoir membrane 42 may generally be used for removing liquid from the reservoir volume 6. The reservoir membrane 42 is shown in Fig. 6. In Fig. 5, two supply ports 35 are illustrated, preferably one used for sampling and / or harvesting and one used for supplying media, agents or the like, are shown from the outside. It may therefore be mentioned that the supply ports 35 or a supply port 35 may serve as a transfer port for adding and / or removing a liquid in general. The reservoir membrane 42 is preferably located below these supply ports 35 and above an also shown reservoir waste port 43 for draining liquid medium filtered by the reservoir membrane 42. The vessel 1 may also comprise tubes connected to the supply port 35 or supply ports 35 and / or the reservoir waste port 43 and preferably comprises a first tube organizer 44 holding these three tubes. The reservoir membrane 42 may be used for cultivating the cells in the liquid medium via perfusion in the reservoir volume 6. Generally, the disclosed vessel 1 allows for a high range of volumes and cell densities due to the rocking motion and / or the membrane or membranes. The reservoir membrane 42 may be used for perfusion in the reservoir volume 6, however, it can also be used additionally or alternatively for washing the cells in the reservoir volume 6 and / or for adding media. Preferably, the reservoir membrane 42 forms a bottom boundary of the reservoir volume 6. Alternatively, the reservoir membrane 42 may form a side boundary of the reservoir volume 6. The reservoir membrane 42 may also be curved.

[0300] According to one embodiment, the reservoir 5 comprises a plane bottom, in particular formed by the reservoir membrane 42 and / or the vessel 1 comprises a boundary between the reservoir 5 and the casing 4 at which boundary walls of the reservoir 5 and walls of the casing 4 are connected and have different inclinations. Fig. 6 shows the different inclinations of the almost vertical walls of the reservoir 5 and the funnel-shaped lower casing part 37.

[0301] There are several preferred features relating to the details of the design of the vessel 1. It may be the case that the lowest point of the casing 4 is the highest point of the reservoir 5, and / or, that the main volume 7 is located fully above the reservoir volume 6, and / or that the boundary between the reservoir volume 6 and the main volume 7 is a horizontal plane, and / or, that the main volume 7 and the reservoir volume 6 are formed by different compartments of the vessel 1. This last feature is especially relevant. The reservoir 5 is one way of forming these different compartments, enabling the high range of different volumes. However, other ways of forming the compartments are also possible and in combination with other features proposed herein may be relevant.

[0302] It may further be the case that the reservoir 5 and the casing 4 have a visible discontinuous boundary, and / or, that the reservoir 5 is bathtub-sha ed and / or the lowercasing part 37 is funnel shaped, and / or, that the lower casing part 37 and / or the reservoir 5 comprise a, in particular strictly, monotonically increasing inner area with increasing height.

[0303] Additionally or alternatively, the inclination of the walls of the lower casing part 45 may always be lower than the highest inclination of the reservoir walls 46, and / or, the inclination of the walls of the lower casing part 45 may be less than 70°, preferably less than 50°, more preferably less than 45°, and / or, the inclination of the reservoir walls 46 may reach a value of 90° at some point, and / or, the upper casing part 36 comprises a, in particular strictly, monotonically decreasing inner area with increasing height. A volume defined by the upper casing part 36 may not be used for containing the liquid medium with the cells, but possibly for condensate, in one embodiment.

[0304] Figure 4 shows that tubes may also be connected to the gas inlet port 8 and / or the gas outlet port 9. Additionally or alternatively, a further gas port 47, may be present, possibly also as the only gas port. A second tube organizer 48 may be present on the top of the vessel l and may hold the tubes connected to the supply port 35 or supply ports 35 and / or the reservoir waste port 43 and / or the further gas port 47.

[0305] It is preferably the case that the reservoir 5 comprises at least one sensor element 16 for measuring a property of the cells and / or the liquid medium in the reservoir volume 6. Preferably, the sensor element 16 is arranged above the reservoir membrane 42.

[0306] It may be the case that the reservoir 5 is a single molded piece, and / or, that the reservoir 5 comprises a material that is optically transparent with a sensor element 16, in particular a dissolved oxygen sensor element 16 and / or a pH sensor element 16 mounted on the inside of the reservoir 5 and readable through the reservoir 5. Preferably, a reservoir wall 46 is thinned at a location of the sensor element 16. The thinning reduces an attenuation of the optical signal.

[0307] According to one embodiment, the vessel 1 comprises a tube loop 49 leading from the vessel 1 and to the vessel 1, in particular for analysis like cell counting. In general, a sensor may be connected to the tube loop 49. In particular, a pH sensor and / or a dissolved oxygen sensor may be connected to the tube loop 49. The loop 49 may connect the supply ports 35. Preferably, the loop 49 comprises at least one branch 50. For example, the shown tube leading from the supply port 35 to the supply or towards a loop destination 51 for a sample or the like may comprise a branch 50, preferably towards a pump and / or a sensor and from there return to another branch 50 of a tube connected to the other supply port 35 for example. The loop 49 in a preferred embodiment is a two-way loop 49. Then the loop 49 goes from one port, in particular through a branch 50, to a loop destination 51, e.g. a sensor. From there, the loop 49 goes on to an air source, in particular the further gas port 47. A sample drawn through the loop 49 is however not routed through the whole loop 49 but instead to the loop destination 51 and back. The part of the loop 49 not used for the liquid is then used for air to push the liquid back and allow for the liquid to flow forward. Schematically Fig. 4 also shows supply and waste bags, pumps and valves. In one embodiment that is not shown, the loop 49 is branch-free. A loop 49 without a branch 50 has an increased utility however necessitates further ports in the vessel 1. It is preferably the case that a volume of the liquid medium in the vessel 1 is actively changed by more than just an addition of the agent. Preferably, a volume of the liquid medium is increased between the start of the cultivation procedure and the end of the cultivation procedure by at least 10 %, preferably at least 50 %, more preferably at least 100 %, more preferably at least 200 %, more preferably at least 300 %, more preferably at least 400 %, more preferably at least 500 %, more preferably at least 600 %, more preferably at least 700 %, more preferably at least 800 %, more preferably at least 900 %. Additionally or alternatively, the volume of the liquid medium inside the vessel 1 may be at most 100 ml, preferably at most 80 ml, more preferably at most 50 ml, more preferably at most 25 ml, more preferably at most 15 ml, and / or at least 5 ml, at the start of the cultivation procedure and / or at least 100 ml, preferably at least 300 ml, more preferably at least 500 ml, more preferably at least 1 1, at the end of the cultivation procedure. It is also possible that the system 2 performs a volume decrease of at least 10 %, preferably at least 30 %, more preferably at least 50 %, on the liquid medium between the start of the cultivation procedure and the end of the cultivation procedure. The volume decrease may be performed through the main membrane 40 and / or the reservoir membrane 42 and / or through a sampling port (e.g. one of the supply ports 35) or by other means.

[0308] Generally, activation and / or genetic modification and / or expansion may be performed in the vessel 1. It is in particular possible that activation is performed with one of the named volumes below 100 ml. The volume may be increased by one of the named percentages for genetic modification by adding a feed medium for example and in particular automatically.

[0309] In particular in combination with the rocking motion it is possible to have a wide range of viable cell counts, for example, the viable cell count of the cell culture between the start of the unit operation and the end of the expansion may vary between at least 10xl06viable cells per ml and 20xl06viable cells per ml, preferably between 5xl06viable cells per ml and 25xl06viable cells per ml, more preferably between lxlO6viable cells per ml and 30xl06viable cells per ml.

[0310] A further embodiment of the vessel 1 is illustrated in Fig. 9. As shown therein, the vessel 1 comprising the casing 4 and reservoir 5 can further comprise a binding unit 101. The binding unit as shown in located at the bottom of the reservoir adjacent to the reservoir membrane 42. The binding unit typically comprises a binding matrix (see grey section in 101) which is configured for binding directly or indirectly to the cells for achieving a positive or negative cell selection step. The binding unit 101 may be provided in form on a column as shown in Fig. 9. Alternatively, the binding unit 101 may be provided as a floating unit (not shown), wherein the binding unit 101 may float within the fluid present in the vessel 1. The binding unit 101 when provided as a column may be directly provided in or adjacent to the reservoir (see Fig. 9). Alternatively, the binding unit 101 may be fluidical ly connected to the vessel 1, preferably to the reservoir 5, such as via a tubing (not shown). The binding unit 101 as shown in Fig. 9 is located adjacent to the reservoir membrane 42. An opening in the reservoir 5 bottom may be provided such that liquid medium can enter the binding unit 101.

[0311] The binding unit 101 as shown in Fig. 9 may comprise may be is by an external loop f I uid ically connected to the reservoir 5 and / or a waste port, preferably both. The external loop may be provided by tubes, which allow for transport of the fluid exiting or entering the binding unit. Such tube preferably comprises a valve as shown in Fig. 9. The external loop comprises at least one valve, preferably one valve for controlling the fluid flow between the binding unit 101 and the waste port and another valve controlled the fluid flow between the reservoir 5 and the waste port, preferably wherein both valves are for controlling the fl uid flow between the binding unit 101 and the reservoir 5 via the external loop. The waste port is preferably connected to a pump allowing fluid transfer operations. Alternatively, the fluid transfer may be achieved by pressurizing the vessel 1 such that the fluid is pushed through the binding matrix 101. It is also possible that an additional entrance is provided between the reservoir 5 and the binding matrix 101, e.g., when providing the binding matrix 101 fluidically connected to the reservoir 5, e.g. by a tube, such that a further tube is connected that can provide over or under pressure to binding matrix 101 for performing fluid transfer operations.

[0312] Another teaching relates to a system 2 for performing a cultivation procedure for cultivating cells in a liquid medium, wherein the system 2 comprises a vessel 1 as provided. The vessel 1 may comprise one or several of the described features. The system 2 as shown in Fig. 1 may comprise an area for processing the cells 52 and a storage area 53.

[0313] According to one embodiment it is proposed that the system 2 comprises a rocker platform 10. The vessel 1 is attached to the rocker platform 10 such that the rocker platform 10 can pivot the vessel 1. The rocker platform 10 can be seen in Fig. 1. The rocker platform 10 may be designed to provide rocking motion. The rocking motion may be uniform or varied in rock angle and speed through its motion. The rocking motion might vary in the course of the cultivation procedure, in particular in dependency from the volume of the liquid medium in the vessel 1. Here, the frequency and / or the angle may be varied. It may be the case that the system 2 comprises a transport mechanism, for example a movable tray 54 mounted on rails (located outside the visible area on the right side in fig. 1) or a robotic arm, for automatically moving the rocker platform 10. At the storage area 53, electrical energy and / or gas may be provided for the rocker platform 10. The rocker platform 10 may comprise an energy source like a battery. The rocker platform 10 may be connected to a gas supply via a plug-connection.

[0314] It is possible, as can be seen in Fig. 1, that the rocker platform 10 comprises a tray 3. The tray 3 may comprise a reservoir recess 55 for receiving the reservoir 5 of the vessel 1. The reservoir recess 55 may determine the orientation of the vessel 1 with regard to the rocker platform 10, particularly the tray 3. It is particularly preferred that the rocker platform 10, particularly the tray 3 and the reservoir recess 55, is designed such that a longitudinal extension 23 of the reservoir 5 is orientated transversely with regard to an axis of rotation, wherein the vessel 1 is moved, in particular repeatedly tilted, around the axis of rotation during the rocking motion, like it is the case in Fig. 1. The tray 3 may comprise alignment elements 56, like pins or the like, for alignment of the casing 4 on the tray 3, as can be seen in Fig. 1. The alignment elements 56 might be designed such that the casing 4 can be fixed on the tray 3. The casing 4 may comprise alignment counterpart elements 57, like holes or the like, for interacting with the alignment elements 56 of the tray 3. The rocker platform 10 may be part of a bioreactor module 58, which is shown in Fig. 1, too. The bioreactor module 58 carries the vessel 1, the rocker platform 10, possibly a control unit, possibly a media bag and / or a waste bag and preferably has a lid 59 for protecting the vessel 1 during transport.

[0315] The inner area described above is looked at when the casing 4 is fully expanded if collapsible. The lower casing part 37 is preferably always fully expanded when the vessel 1 is mounted, here via the alignment elements 56 and alignment counterpart elements 57. Therefore, preferably, the reservoir 5 is hanging freely.

[0316] Regarding the tray 3, it is possible that the tray 3 com rises a tray platform 60, on which the vessel 1, in particular the casing 4, is attached. The tray platform 60 is preferably designed ramp-like (Fig. 1) or funnel-like to support the shape of the vessel 1, in particular the casing 4, preferably the lower casing part 37, in the course of the cultivation procedure. As a result, the liquid medium in the main volume 7 is gravitating to the reservoir volume 6, particularly during the preliminary cultivation phase and harvesting.

[0317] Regarding the tray 3 it is additionally or alternatively possible that the tray 3, particularly the tray platform 60, is designed such that the casing 4, particularly the lower casing part 37, is brought into a funnel-like shape, preferably such that liquid medium containing cells can gravitate from an inner surface of the lowercasing part 37 in the main volume 7 to the reservoir volume 6, when the vessel 1, particularly the casing 4, is attached to the tray 3, particularly the tray platform 60.

[0318] Additionally or alternatively, and as already explained before, the system 2 comprises a sensor unit 17. The sensor unit 17 may comprise a sensor, particularly a pH sensor and / or a dissolved oxygen sensor and / or a biomass sensor. With the biomass sensor, the biomass in the vessel 1 can be measured, particularly the viable cell concentration can be measured.

[0319] The sensor unit 17 is connected to the vessel 1 via a sensor unit connector 20 of the sensor arrangement 15. The sensor unit connector 20 is preferably designed such that the sensor unit 17 is connected to the vessel 1 in an assembly movement of the vessel 1. The vessel 1 might be attached to the rocker platform 10 in the course of the assembly movement. For example, the sensor unit connector 20 may be designed as a plug-in connector and the sensor unit 17 is connected via a plug-in connection movement. It is generally possible that a connection is provided by using spring-loaded pins, in particular for automatically connecting the sensor arrangement 15 and / or the further sensor arrangement 26. Thespring-loaded pins might be part of the vessel 1 and / or particularly part of the system 2, preferably part of the rocker platform 10. Preferably the system 2 comprises a further sensor unit 28, in particular a flow velocity sensor, a pH-sensor, a dissolved oxygen-concentration sensor, etc.

[0320] The system 2 may comprise a control unit. The control unit may be part of the bioreactor module 58. The control unit might be configured to control the sensor unit 17 and / or the further sensor unit 28 of the system 2. Here it is particularly preferred, if the measurement of the properties of the cells and / or the liquid medium is synchronized with the rocking motion. For example, in the growing procedure, particularly in the preliminary phase, the volume in the vessel 1 is relatively small and mainly located in the reservoir volume 6. The vessel 1 is moved in a rocking motion by the rocker platform 10. The measurement of the concentration of the cells by the sensor unit 17 and / or the sensor element 16 or sensor elements 16 of the system 2 is synchronized such that the measurement takes place, if the liquid medium containing the cells is mainly in the sensor section 13. This may improve measurement accuracy. It is also possible that the rocking motion is paused for a certain time period to enable accurate measurements by the sensor unit 17 and / or the sensor element 16 or sensor elements 16 as well as the further sensor unit 28 and / or the further sensor element 29 or further sensor elements 29.

[0321] Furthermore, it is possible that the system 2 comprises a heating arrangement. The vessel 1 is heated by the heating arrangement such that the surrounding area of the vessel 1, in particular of the lower casing part 37 and the upper casing part 36 and / or the reservoir 5, is heated. The heating arrangement may be used in combination with the lid 59. The heating arrangement might be designed such that a heating volume, which, in particular fully, surrounds the vessel 1, is heated evenly. It is also possible that the heating arrangement is designed such that a temperature gradient is adjusted in the heating volume. For example, the upper part of the heating volume might be heated to a higher temperature than the lower part of the heating volume with regard to the horizontal plane. Thus, evaporation and / or condensation issues might be further minimized.

[0322] Another teaching relates to a method for cultivating cells in a liquid medium using a vessel 1 as provided. The vessel 1 may comprise one or several of the described features. The method may be performed by using a system 2, in particular a rocking motion bioreactor, as provided. The method might be a cultivation procedure. All steps described above as performable by the vessel 1 or system 2 may be steps of the proposed method.

[0323] The method may comprise a preliminary cultivation phase, in which liquid medium containing cells is contained in a reservoir volume 6 of the vessel 1 such that cells can be cultivated under certain conditions, and a subsequent cultivation phase, in which liquid medium containing cells is contained in the reservoir volume 6 and in a main volume 7 of the vessel 1 such that cells can be cultivated under certain conditions. The method may comprise that the vessel 1 is moved in a rocking motion, which is in particular performed by a rocker platform 10. Preferably, the subsequent cultivation phase comprises a perfusion procedure, which is particularly performed at least partly via a perfusion arrangement 41 of the vessel 1.

[0324] According to one embodiment it is proposed, that the cells are dedicated for cell and / or gene therapy. Preferably, the cells are immune cells.

[0325] The term "immune cells'" generally refers to different types of white blood cells. Hence, the term "immune cells" includes a variety of cells, for example, but not limited to dendritic cells, T lymphocytes, also referred to as T cells, B lymphocytes, natural killer cells, macrophages or the like. Immune cells may also include subtypes of immune cells, for example tumor-infiltrating lymphocytes or different types of T cells. Subtypes of a certain type of immune cells may be classified based on the type of antigen present at the cell surface. Hence, the term immune cells may for example refer to T cells comprising the surface antigen CD4 ("CD4+ T cells"). Preferably, the cells are genetically modified T cells or are genetically modified to become genetically modified T cells. Preferably, the T cells are genetically modified to express a chimeric antigen receptor (CAR). Consequently, the term "CAR-T cells" describes T cells that have been genetically modified to express a CAR. Hence, preferably, the genetically modified T cells are genetically modified CAR-T cells.

[0326] With regard to the disclosed method, it may be the case that air drawn from the vessel 1, in particular via the further gas port 47, is used to remove liquid residues from one or more tubes attached to the vessel 1. Blowing airthrough the tubes is an easy way to remove remaining agents or liquid medium from a tube. Using the air in the vessel 1 ensures sterility and removes the need for an external gas supply in some stages. The system 2 may be adapted, in particular in software and hardware, to use air drawn from the vessel 1 to remove liquid residues from one or more tubes attached to the vessel 1. This feature is also relevant regardless of the pre-shaped reservoir 5. The air may be moved through the loop 49 and / or one or more branches 50 of the tubes.

[0327] Items according to the present disclosure

[0328] The following items provide further advantageous embodiments of the present disclosure:

[0329] 1. Vessel for cultivating cells in a liquid medium in a cultivation procedure, wherein the cultivation procedure comprises a preliminary cultivation phase, in which the cells are preliminarily cultivated, and a subsequent cultivation phase, in which the cells are subsequently cultivated, wherein the vessel (1) comprises a casing (4) and a reservoir (5), wherein the reservoir (5) defines a reservoir volume (6) for receiving the liquid medium in the preliminary cultivation phase, wherein the casing (4) defines a main volume (7) for receiving the liquid medium in the subsequent cultivation phase, wherein the reservoir volume (6) and the main volume (7) are adjacently arranged and are fluidically connected for fluidical exchange, characterized in that the reservoir (5) is a pre-shaped reservoir (5).

[0330] 2. Vessel according to item 1, characterized in that the reservoir (5) comprises a harvesting connection arrangement (30) for draining the cells from the reservoir volume (6), preferably, that the reservoir volume (6) comprises a harvesting location (31) and that the harvesting connection arrangement (30) comprises a harvesting port (32) for draining the cells in the liquid medium at the harvesting location (31), which is fluidically connected to the reservoir volume (6), more preferably, that the harvesting location (31) is a local minimum of the reservoir (5) or reservoir volume (6) with regard to the horizontal plane.

[0331] 3. Vessel according to item 1 or 2, characterized in that the reservoir (5) comprises a supply connection arrangement (33) for fluidically connecting a supply to the reservoir volume (6), preferably, that the reservoir volume (6) comprises a supply location (34) and that the supply connection arrangement (33) comprises a supply port (35) for supplying liquid medium and / or cells and / or chemicals, etc. at the supply location (34), which is fluidically connected to the reservoir volume (6).

[0332] 4. Vessel according to one of the preceding items, characterized in that the casing (4) comprises an upper casing part (36) and a lower casing part (37) and that the reservoir (5) is attached to the lower casing part (37), preferably, that the reservoir (5) is attached to the lower casing part (37) by welding, or, that the reservoir (5) is formed integrally with the lower casing part (37), in particular by injection molding and / or thermoforming.

[0333] 5. Vessel according to one of the preceding items, characterized in that the vessel (1) comprises a main membrane (40), in particular for cultivating the cells in the liquid medium via perfusion in the main volume (7) in the subsequent cultivation phase, preferably, that the main membrane (40) is arranged at the lower casing part (37), in particular surrounding the reservoir (5) or adjacently to the reservoir (5).

[0334] 6. Vessel according to one of the preceding items, characterized in that the vessel (1) is singleuse and pre-sterilized such that the casing (4) and the reservoir (5) and the main volume (7) and the reservoir volume (6) are sterile.

[0335] 7. Vessel according to one of the preceding items, characterized in that the reservoir (5) comprises a reservoir membrane (42) forming a boundary of the reservoir volume (6), preferably for cultivating the cells in the liquid medium via perfusion in the reservoir volume (6) and / or for washing the cells in the reservoir volume (6), preferably, that the reservoir membrane (42) forms a bottom boundary or a side boundary of the reservoir volume (6).

[0336] 8. Vessel according to one of the preceding items, characterized in that the reservoir (5) comprises a plane bottom, in particular formed by the reservoir membrane (42), and / or, that the vessel (1) comprises a boundary between the reservoir (5) and the casing (4) at which boundary walls of the reservoir (5) and walls of the casing (4) are connected and have different inclinations.

[0337] 9. Vessel according to one of the preceding items, characterized in that the reservoir (5) comprises at least one sensor element (16) for measuring a property of the cells and / or the liquid medium in the reservoir volume (6), preferably, that the sensor element (16) is arranged above the reservoir membrane (42).

[0338] 10. Vessel according to one of the preceding items, characterized in that the reservoir (5) is a single molded piece, and / or, that the reservoir (5) comprises a material that is optically transparent with a sensor element (16), in particular a dissolved oxygen sensor element (16) and / or a pH sensor element (16) mounted on the inside of the reservoir (5) and readable through the reservoir (5), preferably, that a reservoir wall (46) is thinned at a location of the sensor element (16).

[0339] 11. Vessel according to one of the preceding items, characterized in that the vessel (1) comprises a tube loop (49) leading from the vessel (1) and to the vessel (1), in particular for analysis like cell counting, preferably, that the loop (49) comprises at least one branch (50).

[0340] 12. System for performing a cultivation procedure for cultivating cells in a liquid medium, wherein the system (2) comprises a vessel (1) according to one of items 1 to 11.

[0341] 13. System according to item 11, characterized in that the system (2) com rises a rocker platform (10) and that the vessel (1) is attached to the rocker platform (10) such that the rocker platform (10) can pivot the vessel (1), and / or, that the system (2) comprises a sensor unit (17) and that the sensor unit (17) is connected to the vessel (1) via the sensor unit connector (20), preferably, that the sensor unit (17) is connected to the vessel (1) in an assembly movement of the vessel (1), in which the vessel (1) is attached to the rocker platform (10), and / or, that the system (2) comprises a heating arrangement, wherein the vessel (1) is heated by the heating arrangement such that the surrounding area of the vessel (1), in particular of the lower casing part (37) and the upper casing part (36) and / or the reservoir (5), is heated.

[0342] 14. Method for cultivating cells in a liquid medium using a vessel (1) according to one of the items

[0343] I to 11. 15. Method according to item 14, characterized in that the cells are dedicated for cell and gene therapy, preferably, that the cells are genetically modified T cells or are genetically modified to become genetically modified T cells, more preferably, that the genetically modified T cells are genetically modified CAR-T cells.

[0344] Throughout the description, where methods, compositions or uses are described as having, including, or comprising specific components orsteps, it is contemplated that, additionally, there are methods, compositions or uses of the present invention that consist essentially of, or consist of, the recited components or steps.

[0345] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or com onents, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0346] Terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0347] The use of the term "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0348] Where the use of the term "about" or "approximately" is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

[0349] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or"). The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0350] All citations are hereby incorporated by reference. All individual embodiments and aspects as disclosed herein can be combined with each other within the framework and context of the present disclosure. It will be understood that the embodiments disclosed herein are only exemplary, and that any feature presented for a particular exemplary embodiment may be used with the present disclosure on its own or in combination with any feature presented for the same or another particular exemplary embodiment and / or in combination with any other feature not mentioned. It will further be understood that any feature presented for an example embodiment in a particular category may also be used in a corresponding manner in an example embodiment of any other category.

[0351] EXAMPLES

[0352] It should be understood that the following example is for illustrative purpose only and not to be construed as limiting this invention in any manner. The following example demonstrates that the vessel and system according to the present disclosure can be advantageously used for performing certain bioprocesses on the cell culture, specifically on an immune cell culture comprising T cells. In particular, it is shown that T cells can be transferred into the vessel. Afterwards, the cells are activated and expanded in the very same vessel, allowing for simple, precise and robust production of a cell therapy product.

[0353] Example 1

[0354] Peripheral blood mononuclear cells (PBMCs) were thawed and isolated using CD3+ StemCell Technologies Kit according to the manufacturer's instructions. Part of the cells were activated in flask for reference (herein referred to as "BRO2") according to the manufacturer's protocols and part were used for performing the methods according to the present disclosure (herein referred to as "BRO3"). In addition, cells derived from a different donor were activated by the method according to the present disclosure (herein referred to as "BRO1").

[0355] In BRO 1 and BRO3, cells were inoculated and activated in the cell vessel mounted on a bioreactor module (50mL at a cell density of 1.43xlO6 / mL). In BRO2, cells were activated in T- flask at 1.43xl06(in 20mL).

[0356] For culture according to the present disclosure (BRO 1 / BRO3), cells were shaken for 1 h to ensure that the added activation reagent and cell culture were evenly uniform (rate 16rpm angle- 4 / +4). The fluidic lines were primed with media 6 mL media to ensure volume was representative. After 24 h, cell activation was completed and the cells were rocked for 30 min to ensure homogenous distribution (rate: 16 rpm angle-4 / +4), then returned to static position. After 48h, cells were rocked at a rate of 16 rpm angle-4 / +4.

[0357] Post 65 h of activation the cells went through three cycles of perfusion to wash off the activation reagent (0.34mL / min). For this purpose, the culture liquid comprising left over activation reagent was retrieved as permeate through a membrane comprised in the cell vessel (here the reservoir of the cell vessel). Afterwards, fresh cell culture medium was added through the fluidic lines. This process was accordingly repeated until the activation reagent has been essentially completely removed. From day 3 on, cell density was set to lxlO6 / mL by dilution and the cells were cultured in the cell vessel up to day 7 (BRO2, BRO3) or up to day 6 (BRO1).

[0358] On day 6 or 7, respectively, cell density was set to lxlO6 / mL again by dilution with fresh cell culture medium and the cells were cultured in the cell vessel for another 2 h interval. Then, perfusion started and carried on until day 13. This was advantageously done using the reservoir membrane and / or the main membrane disclosed herein. At the end of day 13, the cells were harvested.

[0359] As shown in Fig. 6, BRO3 led not only to a larger total number of cells compared to BRO2 (p=0.0002, graph 1) - which would be expected due to the different starting volumes - but also to a higher daily fold expansion. Hence, by applying the method according to the present disclosure and performing a step of expanding the cells, greater cell numbers were achieved in shorter time, allowing for more efficient production of activated T cells, and thus better manufacturing of a cell therapy product.

[0360] In addition, as shown in Fig. 7, the expansion of BRO3 had higher a viable cell density (VCD) compared to BRO2 (p=0.01 graph 1). No significant difference was seen in the VCD during-the batch culture (p=0.84), but a significant difference was observed the moment perfusion culture was initiated. Hence, by applying the method according to the present disclosure and performing a step of expanding the cells, not only higher cell density but also more viable cells were obtained. Thus, not only was production more efficient with higher yields but also of higher quality, which is an important parameter in the manufacture of a cell therapy product.

[0361] As shown in Fig. 10, the expansion of BRO 1 and BRO3 led to comparable cell numbers (graph 1) and fold expansions (graph 2), with both experiments leading to an expansion of above 300-fold. Cell Viability was also measured during the expansion period (graph 3). Following cell activation between days 0 and 3, a marked decline in viability was observed, reaching approximately 50% in both runs. However, from day 3 onward, cell viability showed a recovery trend, progressively increasing until stabilizing at around 95% from day 6 onwards, where it remained consistent for the duration of the observation period. Assessing the activation count (graph 4 and 5) at the end of the activation period showed that more than 80% CD4+and more than 80% CD8+T cells were activated in each run.

[0362] The example shows that the method according to the present disclosure shows improved results for cell activation and expansion over known flask activation protocols. The results obtained by the method according to the present invention are consistent over different donors, and result in a high percentage of viable, activated CD4+and CD8+T cells.

[0363] Example 2

[0364] On Day 0, CD3+ T cells were inoculated in the cell vessel at approximately 1.43 million cells / mL in 50mL (total of -70 million cells) using Nutri-T media (4Cell® Nutri-T GM P PRF, Biological Industries), along with IL-2 (lOOlU / mL) and TransAct (1:100 dilution) for in-bag T cell activation. During the activation process, the cell vessel was static for the first -60 hours, with intermittent rocking at the 24 and 48 hours. At -60 hours, the activation reagents were automatically washed out of the cell vessel via perfusion media exchange. On day 3, the media within the bioreactor was reduced to achieve a cell concentration of -3 million cells / mL, after which the transfection reagents (GFP-mRNA liposomes) were introduced into the cell vessel. The system was static for the first 4 hours of transfection, after which media was added, to achieve a cell concentration of 0.75 million cells / mL. The system was rocked overnight, and after -8 hours, the transfection reagents were washed out of the cell vessel via perfusion media exchange. From days 4-11 the cells underwent the following expansion process: From days 4-7 media was added daily via fed- batch processes to achieve a cell concentration of 1 million cel Is / mL until IL volume was reached in the cell vessel, from days 7-9 media was exchanged via perfusion processes at 0.5 Vessel Volumes per Day (VVD), and at l VVD from days 9- l l. Finally, on day 11, the cells were harvested from the cell vessel. The following analytics were performed: flow cytometry for transfection efficiency and cell phenotyping, Nucleocounterfor viability and cell counts and Cedex Bio HT for metabolite analysis (glucose, lactate, LDH, ammonia).

[0365] A transfection efficiency of -60% 1 day post-transfection was achieved, and a 70-fold expansion from 70 million to 5 billion CD3+ T cells over 11 days, with a final cell viability of -94%.

[0366] Example 3 Peripheral blood mononuclear cells (PBMCs) from a single donor were thawed and isolated using CD3+ StemCell Technologies Kit according to the manufacturer's instructions. Prior to and after T cell isolation, an aliquot was aspirated and subjected to flow cytometric analysis of immune cell markers CD4 and CD8. Part of the isolated cells were activated in flask for reference (herein referred to as "Flask") according to the manufacturer's protocols and part were used for performing the method according to the present disclosure (herein referred to as "BRI" and "BR2").

[0367] In BR I and BR2, the cells were inoculated and activated in the cell vessel mounted on a bioreactor module (50mL at a cell density of 1.43xlCF / mL). In Flask, cells were activated in a T- flask at 1.43xl06(in 20mL).

[0368] For culture according to the present disclosure (BR1 / BR2), cells were shaken for 1 h to ensure that the added activation reagent and cell culture were evenly uniform (rate 16rpm angle-4 / +4). The fluidic lines were primed with media 6 ml_ media to ensure volume was representative. After 24 h, cell activation was completed and the cells were rocked for 30 min to ensure homogenous distribution (rate: 16 rpm angle-4 / +4), then returned to static position. After 48h, cells were rocked at a rate of 16 rpm angle-4 / +4.

[0369] After 65 h of activation, an aliquot was taken from each culture for flow cytometric analysis of immune cell markers. In the culture, the cells went through three cycles of perfusion to wash off the activation reagent (0.34mL / min). For this purpose, the culture liquid comprising left over activation reagent was retrieved as permeate through a membrane comprised in the cell vessel (here the reservoir of the cell vessel). Afterwards, fresh cell culture medium was added through the fluidic lines. This process was accordingly repeated until the activation reagent has been essentially completely removed. From day 3 on, cell density was set to lxlO6 / mL by dilution and the cells were cultured in the cell vessel up to day 6.

[0370] On day 6, cell density was set to lxlO6 / mL again by dilution with fresh cell culture medium. At this stage, aliquots from all cultures were subjected to immunostaining and flow cytometry analysis for immune cell markers. The Flask culture was discontinued at this point, and the BRI and BR2 cells were cultured in the cell vessel for another 2 h interval. Then, perfusion culture was initiated. From days 6-8 media was exchanged via perfusion processes at 0.5 Vessel Volumes per Day (VVD), and at 1 VVD from days 8- 12. On day 12, the cells were harvested from the cell vessel, and aliquots from both cultures were subjected to immunostaining and flow cytometry analysis. Fig. 11 shows that the percentages of immune cells of each kind (CD4+, CD8+, CD47CD8", CD4+ / CD8+) were almost identical between flask-activated and bioreactor-activated cells, indicating that the method according to the present invention does not introduce any specific bias in terms of survival of any particular population of T cells. As expected, CD8+ cells outgrew the CD4+ population by day 12 of culturing, agreeing with common knowledge in this respect. Collectively, the experiments show that T cell activation by the method according to the present invention leads to advantageous increases in activated cell number while keeping with the known ratios of activated T cell populations.

[0371] Example 4

[0372] On Day 0, CD3+ T cells were inoculated in different cell vessels (BRI and BR2) at approximately 1.43 million cells / mL in 50mL each (total of -70 million cells) using Nutri-T media (4Cell® Nutri-T GM P PRF, Biological Industries), along with IL-2 (lOOlU / mL) and TransAct (1:100 dilution) for in-bag T cell activation. During the activation process, the cell vessel was static for the first -60 hours, with intermittent rocking at the 24 and 48 hours. At -60 hours, the activation reagents were automatically washed out of the cell vessel via perfusion media exchange. In BR I, the exchange occurred by perfusing at a rate of 0.5 mL per minute for 10 hours (total exchange: 300 mL) according to a preferred embodiment of the present invention. I n BR2, the exchange occurred by running six exchange cycles, wherein each exchange cycle consisted of first removing 0.5 mL per minute over 50 minutes (total amount removed: 25 mL), adding 25 mL of fresh culture media, and then rocking for 30 minutes (total amount exchanged: 150 mL), according to an embodiment of the present invention.

[0373] In parallel, a control batch was inoculated and activated underthe same conditions in a T25 flask according to the manufacturer's protocols.

[0374] On day 3, the media within the vessel / flask was reduced to achieve a cell concentration of -3 million cells / mL, after which the transfection reagents (GFP-mRNA liposomes, 0.27 pg mRNA per million cells) were introduced into the cell vessel. The vessel was constantly rocked for the first 4 hours of transfection, after which media was added to achieve a cell concentration of 0.75 million cells / mL. The system was rocked overnight, and after -8 hours, the transfection reagents were washed out of the cell vessel via perfusion media exchange. From days 4-7 media was added daily via fed-batch processes to achieve a cell concentration of 1 million cells / mL until IL volume was reached in the cell vessel. On days 0, 4 and 7, flow cytometry for transfection efficiency and cell phenotyping was performed.

[0375] Fig. 12 A shows that the cell vessel BRI contained >80% GFP-expressing cells on day 4, indicating a transfection efficiency of >80% in the cell vessel, which is suitable and preferred for biotechnological production processes. The cell vessel BR2 contained >50% GFP-expressing cells on day 4, which indicates a transfection efficiency of >50% which is adequate for many processes. The control sample of flask-transfected cells contained approx. 100% GFP-positive cells, and the non-transfected negative control (cells in flask) expectedly contained no GFP- positive cells. On day 7, the percentage of GFP-positive cells was determined only for BR I, and showed to be approx. 70%.

[0376] Fig. 12B shows that the percentages of T cell populations (CD4+ / CD8‘, CD47CD8+, CD47CD8", CD47CD8*) were highly similar between the different approaches (BR I, BR2, cells transfected in flask, untransfected cells in flask), further underscoring that the methods of the invention do not adversely affect and particular population of T cells.

[0377] The experiment shows that the methods according to the invention achieve a high transfection efficiency when transfecting directly inside a perfusion vessel of the invention. In certain embodiments, the transfection efficiency can be improved even further.

[0378] Example 5

[0379] CD3+ T cells were inoculated and activated in a bioreactor as described above under Example 2. 24 hours after activating the cells, cells were transduced to express a recombinant chimeric antigen receptor (CAR) by adding Lentivirus and transduction enhancerto the culture (MOI: 10). A non-transduced culture was cultured in parallel. From day 2 until day 7, fresh cell culture media was added daily via fed-batch processes to the culture to achieve a cell concentration of 1 million cells / mL until IL volume was reached in the cell vessel. From days 7-10, media was exchanged via perfusion processes at 1 VVD. Finally at day 10 cells were harvested. Starting on day 4 (i.e., 72 h post transfection), an aliquot was taken daily and analyzed for total cell number and viability. It was found that the total number of cells and the ell viability was comparable between transduced and non-transduced cells, with transduced cells achieving an approx. 180-fold expansion over the entire culture period. Moreover, it was found that approximately 30% of T cells expressed a CAR, and the number of CAR expressing T cells was in ratio with non-CAR expressing cells (see Fig. 13). The example shows that the method according to the present invention allows for the reliable production of high numbers of CAR T cells. The method enables translating from a manual plate culturing setup to a large-scale process which is at least partially automated.

[0380] While the Examples are representative for embodiments of the present disclosure using activation as unit operation and a subsequent expansion step, different further steps or different embodiments, such genetic modification an automated manner can be achieved. Hence, various different sequences of at least one unit operation and an expansion step can be performed.

Claims

CLAIMS1. A vessel for cultivating cells in a liquid medium in a cultivation procedure, wherein the vessel (1) comprises a casing (4) defining a main volume (7), and a reservoir (5) defining a reservoir volume (6), wherein the reservoir volume (6) and the main volume (7) are adjacently arranged and are configured to be fluidically connected for fluidic exchange, characterized in that the reservoir (5) is a pre-shaped reservoir (5).

2. The vessel according to claim 1, wherein the reservoir (5) is attached to the casing (4), preferably to a casing part, more preferably a lower casing part (37), or the reservoir (5) is formed integrally with the casing (4), preferably with a casing part, more preferably with a lower casing part (37).

3. The vessel according to one or more of claim 1 or 2, wherein the vessel (1) comprises at least one filter which is configured to allow liquid to flow through but essentially retain cells.

4. The vessel according to claim 3, wherein the at least one filter is a membrane, such as a planar membrane, a hollow fiber membrane and / or floating membrane.

5. The vessel according to claim 3 or 4, wherein the at least one filter comprises a main membrane (40), and / or a reservoir membrane (42); preferably both of a main membrane (40) and a reservoir membrane (42).

6. The vessel according to claim 5, wherein the main membrane (40) has one or more of the following characteristics: it is arranged at the casing (4), preferably at a casing part, more preferably at the lower casing part (37); it is fluidically connected to the casing (4), preferably wherein fluidic connections between the casing (4) and the main membrane (40) are provided, in particular wherein the fluidic connections are provided by one or more tubes; it surrounds the reservoir, is adjacent to the reservoir (5) and / or floats within the main volume (7), preferably wherein the main membrane is adjacent to the reservoir; it is configured for removing and / or adding liquid through the main membrane (40) but essentially retaining the cells within the main volume (7) or preferably, within the main volume (7) and the reservoir volume (6); and / orit is configured for cultivating the cells in the liquid medium via perfusion in the main volume (7) or preferably, in the main volume (7) and the reservoir volume (6).

7. The vessel according to claim 5 or 6, wherein the reservoir membrane (42) has one or more of the following characteristics: it is arranged at the reservoir (5), preferably the reservoir (5) comprises the reservoir membrane (42); is fluidically connected to the reservoir (5), preferably wherein fluidic connections between the reservoir (5) and the membrane are provided, more preferably wherein the fluidic connections are provided by one or more tubes; it is configured for removing and / or adding liquid through the reservoir membrane (42) but essentially retaining the cells within the reservoir volume (6) and / or the reservoir volume (6) and the main volume (7); it is configured for cultivating the cells in the liquid medium via perfusion in the reservoir volume (6), and / or the reservoir volume (6) and the main volume (7); and / or it is configured for retaining the cells in the reservoir volume (6) when removing a fluid from the reservoir volume (6) and / or the main volume (7), such as when washing the cells in the reservoir volume (6), preferably configured for washing the cells in the reservoir volume (6).

8. The vessel according to one or more of claims 1 to 7, wherein the reservoir (5) comprises at least one sensor element (16) for measuring at least one parameter of the cells and / or the fluid, such as the liquid medium, in the reservoir volume (6); and / or one or more sensor ports configured for allowing a sensor to be inserted.

9. The vessel according to one or more of claims 1 to 8, wherein the reservoir (5) is a single molded piece; comprises a material that is optically transparent with a sensor element (16), in particular a dissolved oxygen sensor element (16) and / or a pH sensor element (16) mounted on the inside of the reservoir (5) and readable through the reservoir (5); and / or comprises a reservoir wall (46) that is thinned at a location of the sensor element ( 16).

10. The vessel according to one or more of claims 1 to 9, wherein the reservoir volume is at most 250 ml, preferably at most 150 ml or 120 ml; and / or the main volume is at most 6 I, preferably at most 3 I or 2 I.

11. A system for cultivating cells in a liquid medium in a cultivation procedure, wherein the system (2) comprises a vessel (1) according to one of claims 1 to 10, preferably the system (2) further comprises:i. a rocker platform (10), preferably wherein the vessel (1) is attached to the rocker platform (10), more preferably such that the rocker platform (10) can pivot the vessel (1); ii. a sensor unit (17), preferably wherein the sensor unit (17) is connected to the vessel (1) via the sensor unit connector (20); and / or iii. a heating arrangement, preferably wherein the vessel (1) is heated by the heating arrangement, in particular such that the surrounding area of the vessel (1) is heated.

12. A method for cultivating cells in a liquid medium and / or for manufacturing a cell therapy product comprising: a) providing cells in a liquid medium, wherein the cells are immune cells, preferably T cells; b) cultivating the cells using a vessel (1) according to one or more of claims 1 to 10 and / or a system according to claim 11.

13. The method according to one or more of claim 12, wherein step (b) comprises(b.l) a preliminary cultivation phase, in which the cells are preliminarily cultivated, preferably wherein cells are predominantly cultivated in the reservoir (5); and(b.2) a subsequent cultivation phase, in which the cells are subsequently cultivated preferably wherein cells are cultivated in the reservoir (5) and the casing (4).

14. The method according to claim 12 or 13, wherein the method further comprises one or more of the following steps: optionally, selecting cells, in particular by binding at least a fraction of the cells expressing a ligand, preferably selecting a subpopulation of T cells; activating cells, preferably activating T cells; transducing cells, preferably transducing T cells; transfecting cells, preferably transfecting T cells; and / or expanding cells, preferably T cells, more preferably genetically modified T cells; preferably, wherein the method comprises at least two, three or four of the above-mentioned steps.

15. The method according to one or more of claims 12 to 14, wherein the method further comprises after step b) formulating the expanded cell culture into a media suitable for being injected into a patient and / or transferring the expanded cell culture from the cell vessel (6) into a harvesting vessel.

16. Use of the vessel according to one of claims 1 to 10 and / or system according to claim 11 for manufacturing a cell therapy product, preferably wherein manufacturing comprises at least one of the following:optionally, selecting cells, in particular by binding at least a fraction of the cells expressing a ligand, preferably selecting a subpopulation of T cells; activating cells, preferably activating T cells; transducing cells, preferably transducing T cells; transfecting cells, preferably transfecting T cells; and / or expanding cells, preferably T cells, more preferably genetically modified T cells.

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