WO2022075842A1

WO2022075842A1 - Cell culture bag, bioreactor and cell filtration device

Info

Publication number: WO2022075842A1
Application number: PCT/NL2021/050603
Authority: WO
Inventors: Emerentius Gerardus ROOSLOOT; Marijn Anna Wilhelmina DRIESSEN; Rudolf Hendrikus Jozef DAS; Michiel Johannes Antonius JANNINK
Original assignee: Scinus Holding B.V.
Priority date: 2020-10-05
Filing date: 2021-10-05
Publication date: 2022-04-14
Also published as: EP4225885A1; JP2023543546A

Abstract

A cell culture bag for cultivating cells under rocking conditions in a bioreactor system comprises a deformable flexible wall defining an expandable cell culture chamber for containing the cells in a cell culture medium. A filter is arranged inside the culture chamber dividing the culture chamber in a chamber top compartment for containing cell-free culture medium and a chamber bottom compartment for maintaining the cells. The filter comprises a membrane with a pore size configured to allow exchange of nutrients and gasses between the chamber bottom compartment and chamber top compartment while retaining cells in the chamber bottom compartment. The filter is connected to the deformable flexible wall of the cell culture bag via a filter support frame that is configured and arranged to support the filter and mitigate forces acting on the filter when filling and/or expanding and/or rocking the bag.

Description

Title: Cell culture bag, bioreactor and cell filtration device

FIELD OF THE INVENTION

The invention relates to a cell culture bag for culturing cells. The invention moreover relates to a bioreactor for cultivating cells under rocking conditions. The invention further relates to a cell filtration device.

BACKGROUND

Cell culture bags for culturing useful cells for treating diseases and disorders are known to provide suitable cell culture chambers for maintaining and expanding the cells. In particular, cell culture bags are suited for an increase of the volume of the cell culture chamber. The increase in volume of the cell culture chamber allows the cells to be maintained in the same culture chamber with a dedicated and optimized cell number to volume ratio when the number of cells in the cell culture increases during expansion, obviating a need to transfer the cells from one container with a first volume to another container with another volume, while enabling control over the specific environment in the culture chamber, such as maintaining a suitable fluid culture medium with a constant optimal temperature, pH and concentration of dissolved oxygen (DO), during the expansion of the cell culture.

For instance W02015009153A1 describes an expandable cell culture bag suitable for culturing cells wherein the bag comprises a deformable flexible wall defining an expandable cell culture chamber for containing the cells in a cell culture medium. The deformable flexible wall allows easy expansion of a volume of the cell culture chamber by initially maintaining the bag with portions of the deformable flexible wall pinched on top of each other between a support surface, e.g. the top face of a rocker platform for rocking the bag, and a clamping or pinching means, e.g. a roller, positioned above the support surface. By controlled movement of the pinching means the pinched portion of the deformable flexible wall may be released at least in part to allow expansion of the volume of the cell culture chamber as desired. An inlet port and one or more outlet ports are provided in the deformable flexible wall of the cell culture bag to allow exchange of fluids and cells in and out of the cell culture chamber independent of an expansion state of the bag, i.e. a clamped state and minimum volume cell culture chamber or a fully expanded state and maximum volume of the cell culture chamber and all states with corresponding volumes in between. A membrane is provided between the at least one outlet and the culture chamber having a pore size which allows anchorage dependent, or adherent, cells, e.g. human stem cells, to be separated from anchorage independent, or suspension, cells, e.g. certain white blood cells, by anchoring the adherent cells to microcarriers which cannot pass through the pores of the membrane. Thus the expandable cell culture bag is well suited for expanding anchorage dependent, or adherent, cells out of any anchorage dependent cells containing starting cell material including crude biopsy material.

It is an aim of the present invention to provide an expandable cell culture bag that is well suited for expanding cells such as particularly suspension cells. In particular the present invention aims to provide an expandable cell culture bag that enables expanding of suspension cells from a small starting cell culture having a relatively low number of cells to a clinically useable cell culture having an suitable and/or desired amount of cells. It is a further aim of the present invention to provide a bioreactor for cultivating and expanding cells, particularly suspension cells, from a small starting cell culture having a relatively low number of cells to a clinically useable cell culture having an suitable and/or desired amount of cells. Another aim of the present invention is to provide a cell filtration device that enables retaining of cells while allowing exchange of nutrients and gasses from one side of the cell filtration device to an opposite side of the cell filtration device by diffusion.

DESCRIPTION OF THE INVENTION

In a first aspect there is provided a cell culture bag for cultivating cells under rocking conditions in a bioreactor system, the bag comprising a deformable flexible wall defining an expandable cell culture chamber for containing the cells in a cell culture medium, and a filter arranged inside the culture chamber dividing the culture chamber in a chamber top compartment for containing cell -free culture medium and a chamber bottom compartment for maintaining the cells, the chamber top compartment provided with an inlet port for filling the cell culture chamber and the chamber bottom compartment provided with an outlet port for draining of the culture chamber, wherein the filter comprises a membrane with a pore size configured to allow exchange of nutrients and gasses between the chamber bottom compartment and the chamber top compartment while retaining cells in the chamber bottom compartment and wherein the filter is connected to the deformable flexible wall via a filter support frame that is configured and arranged to support the filter and mitigate forces acting on the filter when filling and/or expanding and/or rocking the bag.

The membrane may optionally be configured to prevent any cells from migrating through the membrane from the chamber bottom compartment to the chamber top compartment. For example the membrane may have a pore size small enough for blocking any cells to pass, e.g. pores with a diameter 1 pm or smaller. Preferably at least suspension cells such as certain white blood cells are prevented from passing through the membrane from the chamber bottom compartment to the chamber top compartment. Fluids such as gases or liquids, e.g. cell culture medium, can pass through the pores of the membrane unhindered, for example by perfusion and/or diffusion. Preferably in use of the cell culture bag cell culture nutrients in a cell culture medium may diffuse through the membrane obviating a need for active perfusion of the cell culture medium through the membrane. Possible clogging of the membrane by cells is hereby greatly reduced or prevented. Optionally fresh cell culture medium may be supplied to the chamber top compartment via the inlet port by means of pressurized flow, e.g. using a perfusion device and/or pump. Optionally a chamber top compartment outlet is fluidically connected to the inlet port to enable circulation of the cell culture medium in the chamber top compartment. For example cell culture medium in the chamber top compartment can be pumped out via the outlet port and circulated through an oxygenator to control continuous gas supply to the cell culture medium and re-enter the chamber top compartment through the inlet port. Active or pressurized flow of the cell culture medium from the chamber top compartment to the chamber bottom compartment may be prevented with any exchange of nutrients between the chamber top compartment and chamber bottom compartment being by diffusion.

In order to enable sufficient supply of nutrients to the expanding cell culture in the cell culture chamber, i.e. to the increasing number of cells over time, particularly using mainly or only diffusion, the filter needs to have a sufficiently large filtration surface between the culture chamber bottom compartment and culture chamber top compartment. Accordingly the membrane of the filter preferably extends at least almost fully over a largest cross sectional area of the culture chamber defined by the deformable flexible wall. In particular the membrane of the filter defines a filtration surface which has approximately the same area size as a largest cross sectional area of the culture chamber. The actual area size of the membrane filtration surface thus may depend on a size of the cell culture chamber in the cell culture bag, but practically is preferably at least 150cm², more preferably 200cm² or higher, even more preferably at least 220cm², such as between 220cm² and 230cm², for example 224cm². Such areas in practice enable sufficiently high diffusion rates for membranes with suitable pore sizes, thickness and materials to maintain the growing cell culture with good viability. The membrane may optionally be made of, or comprise a suitable material that allows nutrients, particularly glucose, to pass the membrane by diffusion when provided with a pore size that prevents cells from passing, preferably pores with a diameter of 1pm or less. For example the membrane may be made of, or comprise nylon and/or PVDF. Optionally the membrane may have pores with an average largest diameter or absolute largest diameter of Igm or lower, preferably approximately 0,8jim. Preferably the membrane is a relatively thin layer to allow relatively high diffusion rates. The thickness of the membrane may be taken to be as thin as possible while providing sufficient tear resistance in use, wherein the actual thickness depends on a size of the membrane, material and porosity.

The membrane optionally divides the culture chamber such that the chamber top compartment has a first volume and the chamber bottom compartment has a second volume wherein the second volume preferably is larger than the first volume. Preferably, upon expansion of the cell culture bag the volume ratio of the first volume to the second volume decreases, i.e. the chamber bottom compartment increases more in volume than the chamber top compartment when expanding the cell culture bag. The chamber top compartment preferably has a volume adapted to maintain a sufficient amount of cell culture medium for maintaining the cell culture in the chamber bottom compartment. The chamber bottom compartment preferably has a volume that upon expanding the cell culture bag increases to maintain cells in a suitable cell quantity to volume ratio in the chamber bottom compartment. The cell culture bag when supported on a surface in fully expanded state may for example be pillow-shaped with a bag bottom part and bag top part connected to each other forming a periphery edge, wherein the membrane extends horizontally at a height of the periphery edge to divide the cell culture chamber in the chamber top compartment and the chamber bottom compartment.

In order to provide such membrane with relatively large filtration surface and with appropriate and reliable filtering properties and diffusion rates in use of the expandable cell culture bag the membrane is supported by the filter support frame which mitigates forces acting on the filter in use. Preferably undesired deformation of the membrane, which amongst others could cause the porosity of the membrane to change thereby affecting the filtering properties thereof, is at least almost completely prevented during filling of the cell culture bag and use thereafter. Particularly the membrane, which is preferably provided as a flat horizontal layer extending from one side of the bag to an opposite side of the bag, is prevented by the support frame from bending, folding or otherwise deforming or changing in shape which may lower the reliability of filtration properties of the filter.

The filter support frame is configured to enable an expansion of the cell culture chamber while maintaining desired filtering properties of the membrane, i.e. the membrane provides the same filtering properties in the different expansion states of the cell culture bag. In addition to the filtering properties, also other properties of the membrane, in particular a durability thereof, may be improved by the filter support frame by reducing the forces being exerted on the membrane in use of the cell culture bag.

Optionally the flexible wall of the cell culture bag is made of a polymer material suitable for use with living cells, and preferably comprises one or more of EVE, EVOH, PE and PVC.

In a further aspect the support frame comprises a substantially rigid body made of the same material as the flexible wall. The support frame may comprise a polymer material such as EVE, EVOH, PE and PVC. Alternatively the support frame may comprise or be made of another material. The rigid body may be formed as a skeleton frame extending around a central opening in which the membrane may be provided. The support frame in particular may comprise an inner edge section that is attached to the filter and an outer edge section that is attached to the flexible wall. The support frame thus enables a connection of the filter to the flexible wall of the cell culture bag without having to attach the filter to the flexible wall directly. The cell culture bag may comprise a bag bottom part and bag top part connected to each other forming a periphery edge. Preferably, the outer edge section of the support frame is attached to the flexible wall at the periphery edge. For example the bag bottom part and bag top part may be glued, welded or otherwise adhered to each other at the periphery edge with the outer edge section of the support frame clamped or otherwise secured in between respective portions of the bag bottom part and bag top part. The inner edge section may be glued, welded or otherwise adhered to the filter. The inner edge section of the filter support frame may for example be directly attached to an outer edge portion of the membrane by welding using heat and/or pressure. The inner edge section may alternatively be connected indirectly to the membrane, for example via an intermediate connecting body or means of the filter.

For example in another aspect the filter comprises a stretchable support layer attached to the membrane, which stretchable support layer is configured to allow stretching of the filter. The support layer may be attached to the support frame, particularly welded to the support frame, providing a connection between the support frame and the membrane without having to attach the support frame directly to the membrane. The stretchable support layer is configured to allow some stretching of the support layer. The stretchable support layer is for example elastic and/or made of an elastic material. In an aspect the stretchable support layer is made of a woven material. The stretchable support layer particularly prevents pull and/or stretch forces acting on the membrane. For example when the cell culture bag is brought from an initial small volume of the cell culture chamber into a more expanded or fully expanded cell culture chamber with larger volume any remaining forces caused by the expansion of the flexible wall and not mitigated by the support frame will first act on the stretchable support layer attached to the support frame before having an effect on the membrane. Accordingly the stretchable support layer forms a further protective means reducing or preventing any forces acting on the membrane, thereby increasing a reliability of the membrane in view of filtering properties as well as a durability thereof.

In an aspect the filter comprises an antifolding layer attached to the membrane and configured to protect the membrane against folding. The antifolding layer may be provided on one or both sides of the membrane. For example the antifolding layer may be a rigid body in the filter to strengthen the filter against undesired bending forces acting on the membrane. That is to say, the antifolding layer may prevent bending or folding of the filter in one direction while allowing bending or folding of the filter in another direction. For example the filter in use of the cell culture bag may be bent in one direction in a pinched state of the cell culture bag and the filter may be straight, i.e. extending at least almost fully in one plane, in the expanded state of the cell culture bag. The antifolding layer however acts against any bending or folding of the membrane in another direction.

Preferably the antifolding layer is positioned opposite the filtration surface of the membrane. For example the antifolding layer may be attached against the membrane and extend parallel to the filtration surface of the membrane. In a particular aspect the antifolding layer may be sized to cover at least almost the whole filtration surface of the membrane. In another particular aspect the stretchable support layer may be sized to cover at least almost the whole filtration surface of the membrane. Optionally the antifolding layer comprises a rigid mesh structure. The mesh openings may be sized to have no effect on a diffusion rate of components such as nutrients and growth factors in cell culture medium moving through the membrane. In a further aspect the filter comprises the membrane sandwiched between the antifolding layer and the stretchable support layer. The layers may be adhered to each other forming a three layered body that may be attached to the filter support frame, preferably via the stretchable support layer. For example the three layered body may be positioned inside an opening defined by a skeleton frame of the filter support frame with an outer edge portion of the stretchable support layer being welded to the inner edge section of the filter support. The filter attached to the filter support frame forms a cell filtration device that can be conveniently attached inside the cell culture chamber of the cell culture bag by securing the filter support layer against the flexible wall.

In another aspect there is provided a cell filtration device comprising a filter with a membrane having a pore size configured to allow exchange of nutrients and gasses from one side of the membrane to an opposite side of the membrane by diffusion while retaining cells on one side of the membrane, and comprising a filter support frame to which the filter is connected the support frame configured and arranged to support the filter and mitigate stretching forces acting on the filter against the flexible wall. In particular the cell filtration device may comprise a filter support frame and/or a filter as used in the cell culture bag described herein.

A further aspect is the provision of a bioreactor system for culturing cells comprising a cell culture bag, for example as described herein, for cultivating cells under rocking conditions, wherein the bag comprises a wall defining a cell culture chamber for containing the cells in a cell culture medium, and a filter arranged inside the culture chamber dividing the culture chamber in a chamber top compartment for containing cell-free culture medium and a chamber bottom compartment for maintaining the cells, the chamber top compartment provided with a first inlet port and the chamber bottom compartment provided with a second inlet port, for filling the cell culture chamber, wherein the culture chamber is provided with an outlet port for draining of the culture chamber, wherein the filter comprises a membrane with a pore size configured to allow exchange of nutrients and dissolved gasses between the chamber bottom compartment and chamber top compartment while retaining cells in the chamber bottom compartment, wherein the system comprises a fluid conduit circuit that defines a first culture medium flow path that extends from the outlet port of the cell culture chamber to the first inlet port, and a second culture medium flow path that extends from the outlet port of the cell culture chamber to the second inlet port. This way, the culture medium that is extracted at an outlet of the cell culture bag is reintroduced into the cell culture bag in the chamber top compartment as well as in the bottom compartment, enabling control of the culture medium distribution over the chamber top and chamber bottom compartment. For example, a volume difference and/or pressure difference between the chamber top and chamber bottom compartment could be mitigated by supplying the culture medium to both the chamber top compartment and the chamber bottom compartment. The culture medium may be treated, e.g. filtered, oxygenated, purified, refreshed, and/or provided with nutrients, while the culture medium is transferred along the first culture medium flow path and/or the second culture medium flow path.

A pump can be provided for pumping the culture medium through the fluid conduit circuit. The outlet of the cell culture bag may be formed by an outlet port of the chamber top compartment and/or by the outlet port of the chamber bottom compartment. For example, the outlet of the cell culture bag may be formed by an outlet port of chamber top compartment, wherein culture medium is extracted from the cell culture bag through said outlet port of the chamber top compartment, transported along the first flow path in which the culture medium is treated and reintroduced into the cell culture bag through the inlet port of the chamber top compartment. The first culture medium flow path and the second culture medium flow path may be entirely separate, or may partially coincide. For instance, the second flow path could branch off from the first flow path or vice versa. The first flow path and the second flow path may for example share a common fluid conduit.

The bioreactor system may comprise an oxygenator arranged in the first culture medium flow path and/or the second culture medium flow path for oxygenating the cell culture medium. The oxygenator can for example exchange oxygen and carbon dioxide with the culture medium. The oxygenator is preferably provided in the shared common fluid conduit of the first and second flow paths, wherein the first flow path branches off from the common conduit to deliver oxygenated culture medium directly to the chamber top compartment and the second flow path branches off from the common conduit to deliver oxygenated culture medium directly to the chamber bottom compartment.

A controllable one-way valve may be provided in the second culture medium flow path for controlling the flow of culture medium through the second culture medium flow path. The one-way valve may be controllable by the pump which may be arranged to pump the culture medium from the outlet port to the second inlet port with a flow pressure sufficient to open the one way valve. Optionally a control unit may be provided for controlling the one-way valve, for example based on a measured state of the culture bag, such as a volume of the chamber top compartment and/or chamber bottom compartment or a ratio thereof, a pressure in the chamber top compartment and/or chamber bottom compartment or a ratio thereof, a shape of the filter, or a combination thereof.

In another aspect, a bioreactor system is provided, comprising a cell culture bag, for example as described herein, for cultivating cells under rocking conditions, the bag comprising a wall defining a cell culture chamber for containing the cells in a cell culture medium, and a filter arranged inside the culture chamber dividing the culture chamber in a chamber top compartment for containing cell -free culture medium and a chamber bottom compartment for maintaining the cells, the chamber top compartment provided with a first inlet port for filling the cell culture chamber, wherein the culture chamber is provided with an outlet port for draining of the culture chamber, wherein the filter comprises a membrane with a pore size configured to allow exchange of nutrients and dissolved gases between the chamber bottom compartment and chamber top compartment while retaining cells in the chamber bottom compartment, wherein the system comprises a gas source that is fluidly connected to the inlet port of the chamber top compartment arranged for supplying a gaseous fluid to the chamber top compartment of the cell culture bag. In addition to gases that are dissolved in the culture medium, the gas source may supply the chamber top compartment of the culture bag with a head space of a gaseous fluid, i.e. a fluid in gas phase.

The gas source may for example comprise a source of oxygen gas, that is introduced into the chamber top compartment through the inlet port of the chamber top compartment. The gaseous head space can increase the availability of oxygen for the cells while the cells held in the chamber bottom compartment remain physically separated from the gaseous head space. The liquid culture medium can for instance exchange oxygen and carbon dioxide, wherein the oxygen dissolves in the culture medium and diffuses across the filter from the chamber top compartment to the chamber bottom compartment to be consumed by the cells contained therein. The gas source for example comprises a pressurized gas container.

The gas source is optionally formed by an oxygenator of the bioreactor system. For instance a residual gas stream of the oxygenator, containing gases that have not dissolved in the culture medium during oxygenation, may be fluidly connected to the chamber top compartment to be supplied thereto. To minimize the number of ports of the cell culture bag, the gaseous fluid may be combined with a stream of the liquid culture medium before entering into the culture bag. The gaseous fluid may be passed through a filter, e.g. a particle filter, prior to being combined with the culture medium, to prevent contaminating the culture medium.

A degassing valve may be provided in the first flow path and/or the second flow path for separating a liquid phase and a gas phase from each other. The degassing valve is preferably arranged in a section first flow path and/or the second flow path upstream of the oxygenator.

In an aspect is provided a cell culture bag for cultivating cells under rocking conditions in a bioreactor system, the bag comprising a deformable flexible wall defining an expandable cell culture chamber containing the cells in a liquid cell culture medium, and a filter arranged inside the culture chamber dividing the culture chamber in a chamber top compartment containing cell -free culture medium and a chamber bottom compartment maintaining the cells, the chamber top compartment provided with an inlet port for filling the cell culture chamber and an outlet port for draining of the culture chamber, wherein the filter comprises a membrane with a pore size configured to allow exchange of nutrients and dissolved gases between the chamber bottom compartment and chamber top compartment while retaining cells in the chamber bottom compartment, wherein the chamber top compartment further contains a fluid in gas phase.

A further aspect is the provision of a bioreactor system, e.g. as described herein, for culturing cells comprising a cell culture bag as described herein and an agitator unit arranged for operative coupling with the cell culture bag to agitate the cell culture and the fluid cell medium in the cell culture chamber and a mechanism for pinching the outer wall of the variable volume container to create a partially flattened section and operable for increasing the cell-culture chamber in volume by decreasing the flattened section.

The bioreactor system may be a bioreactor system as described in WO20 15009153A1 however with the cell culture bag being replaced by a cell culture bag as described herein, which cell culture bag is suited to particularly expand a cell culture comprising suspension cells by cultivating the cells in the expanding volume under rocking conditions to maintain an approximately evenly distributed amount of cells in desired concentration in suspension in the cell culture chamber. The cell culture bag may be single use only, or alternatively may be reusable.

The cells to be cultured in the cell culture bag and/or bioreactor system may be any kind of cells, such as bacterial cells, Fungi, yeast cells, plant cells, algae, insect cells, mammahan cells. Preferably the cells are mammalian cells, more preferably human cells, more preferably human suspension cells such as blood cells. The cells may in particular be chosen from stem cells, embryonic cells, red blood cells, various white blood cells such as T-cells, B-cells, and natural killer cells, omnipotent cells, pluripotent cells, and cancer cells. The cells may be for example be primary cells or may be from a cell-line. The cells may be grown in suspension but may also be adherent dependent and require growth for example on scaffolds or microcarriers.

BRIEF DESCRIPTION OF THE FIGURES

These and other aspects of the present invention are hereinafter further elucidated by the appended drawing and the corresponding embodiment, which forms part of the present application. The drawing is not in any way meant to reflect a limitation of the scope of the invention, unless this is clearly and explicitly indicated. In the drawing:

Fig. la shows a perspective view of a cell culture bag in an embodiment according to the invention with a cut-out to image an inside of the bag;

Fig. lb shows a cross sectional view of the culture bag according to fig. la along a vertical plane; Fig. 1c shows a cross sectional detailed view of a cell filtration device as used in the culture bag according to fig. la along a horizontal plane;

Fig. 2 shows a schematic representation of a bioreactor system comprising a cell culture bag.

In this application similar or corresponding features are denoted by similar or corresponding reference signs. The description of the embodiment is not limited to the examples shown in the figures and the reference numbers used in the detailed description and the claims are not intended to limit the description of the embodiments, but are included to elucidate the embodiment by referring to the examples shown in the figures.

DETAILED DESCRIPTION OF THE FIGURES

As shown in figure la, lb, and 1c in an embodiment according to the invention the cell culture bag 1 comprises a deformable flexible wall forming a bag bottom part 2 and a bag top part 3. The bag bottom part 2 and the bag top part 3 are both sheets of a suitable cell culture bag material, preferably polymer material suitable for use with living cells, such as one or more of EVE, EVOH, PE and PVC. The bag bottom part 2 and the bag top part 3 are connected to each other at their circumferential edges, forming a periphery edge 4 of the cell culture bag. An expandable cell culture chamber for containing the cells in a cell culture medium is defined by the deformable flexible wall 2,3. Inside the culture chamber a filter 10 is arranged to divide the culture chamber in a chamber top compartment 5a for containing cell- free culture medium and a chamber bottom compartment 5b for maintaining the cells. The chamber top compartment 5a is provided with an inlet port 6a for filling the cell culture chamber and an outlet 7a for outflow of excess culture medium, maintaining in use a continuous supply of fresh cell culture medium in the chamber top compartment. The chamber bottom compartment 5b is provided with an inlet 6b for supplying cells into the cell culture chamber and an outlet port 7b for draining the culture chamber. The filter 10 comprises a membrane 11 with a pore size configured to allow exchange of nutrients and gasses by diffusion between the chamber top compartment and chamber bottom compartment while retaining cells in the chamber bottom compartment. The filter 10 is connected to the deformable flexible wall 2,3 via a filter support frame 8 that is configured and arranged to support the filter 10 and mitigate forces acting on the filter when filling and/or expanding and/or rocking the bag. The filter 10 and filter support frame 8 form a cell filtration device as described herein. As best seen in fig. 1c the filter comprises the membrane 11 sandwiched between a stretchable support layer 12 made of a woven material attached to the membrane on one side, which stretchable support layer is configured to allow stretching of the filter, and an antifolding layer 13 made of a rigid mesh structure, preferably a sheet of interconnected metal or plastic wires, attached to the membrane on an opposite side, which antifolding layer protects the membrane against folding. The support frame 8 comprises a rigid body having an inner edge welded to the stretchable support layer 12 and an outer edge welded in the periphery edge 4 of the cell culture bag. The cell culture bag is suitable for use in a bioreactor system and for growing cells in an expanding volume, and is particularly suited for expanding suspension cells as described herein. The cell culture bag 1 may be provided with one or more sensors for sensing a parameter of the culture medium. Here three sensors are provided including a biomass sensor, a pH sensor and dissolved oxygen (DO) concentration sensor 20,30,40.

Fig. 2 shows a schematic representation of a bioreactor system comprising a cell culture bag 1. The cell culture bag 1 as shown in Fig. 2 may be similar to the cell culture bag as described in relation to Figs, la, lb, 1c, but it may also be an alternative cell culture bag. Here, the culture bag 1 comprises a bag bottom part 2 and a bag top part 3, that form a culture chamber therebetween. A filter 10 is arranged in the culture chamber, to divide the culture chamber in a chamber top compartment 5a for containing cell-free culture medium and a chamber bottom compartment 5b for maintaining the cells. The filter 10 is supported by filter support frame 8 that is configured and arranged to mitigate forces acting on the filter when filling and/or expanding and/or rocking the bag 1. The chamber top compartment 5a is provided with an inlet port 6a for filling the cell culture chamber with a culture medium and an outlet 7a for draining excess culture medium. The chamber bottom compartment 5b is provided with an inlet port 6b. Here the chamber bottom compartment 5b is not provided with an outlet port, but it may be appreciated that an outlet port may be provided at the chamber bottom compartment.

The bioreactor system further comprises an oxygenator 50 that is provided in a fluid conduit circuit that defines a flow path for the culture medium from the outlet 7a of the bag 1 to the inlet port 6a and/or the inlet 6b, for oxygenating the culture medium. A pump 51 is provided for circulating the culture medium through the fluid conduit circuit via the oxygenator 50, such that the culture medium is continuously refreshed. It will be appreciated that other treatments may be performed on the culture medium, not shown in Fig. 2.

Here the fluid conduit circuit defines a first culture medium flow path 52a that extends from the outlet 7a to the inlet port 6a through the oxygenator, and a second culture medium flow path 52b that extends between the outlet 7a and the inlet 6b. Through the oxygenator 50, the first flow path 52a and the second flow path 52b partly coincide in a common flow path section 53, wherein the first flow path 52a and 52b separate from each other downstream of the oxygenator 50, and downstream of the pump 51. To control the volume/pressure of/in the chamber bottom compartment 5b relative to the chamber top compartment 5a, a controllable valve 54, particularly a controllable one-way valve, is arranged in the second flow path 52b. The valve 54 allows to adjust the flow of culture medium that is delivered to the chamber bottom compartment 5b. When culture medium is circulated through the first flow path 52a only, a volume difference may gradually arise between the chamber top compartment 5a and the chamber bottom compartment 5b causing the filter 10 to deflect towards the chamber bottom compartment 5b, effectively decreasing the volume of the chamber bottom compartment 5b holding cells. This could be mitigated by controlling the flow of culturing medium through the second flow path 52b by means of the valve 54.

The bioreactor system further comprises a gas source for supplying a gaseous fluid to the culture bag 1. Here the gas source is formed by the oxygenator 50, but it will be appreciated that the system may comprise a separate gas source, such as a pressurized gas container. The gas source for example comprises a source of oxygen, carbon dioxide, nitrogen or a mixture thereof that may be supplied in a gas phase to the culture chamber, particularly to the chamber top compartment 5a, to form a head space therein. A boundary between the gaseous fluid in the head space (gas phase) and the culturing medium (liquid phase) is indicated in Fig 2 by a dashed line 55 in the chamber top compartment 5a. The head space could increase the availably of oxygen for the cells maintained in the culture medium. For instance, the head space and the liquid culture medium may exchange oxygen and carbon dioxide at the boundary 55. The filter 10 physically separates the cells contained in the chamber bottom compartment from the gaseous fluid in the head space in the chamber top compartment.

The gaseous fluid may be combined with the culturing medium in the second flow path 52a, to minimize the number of inlet ports of the culture bag. It will be appreciated that a separate gas circuit may be provided for circulating the gaseous fluid separate from the culture medium. For instance, the chamber top compartment 5a may be provided with a separate gas inlet and separate gas outlet, separate from a culture medium inlet and culture medium outlet. A pressure regulator 56 is provided to regulate the pressure of the introduced gaseous fluid. The gaseous fluid may be passed through a particle filter 58 before being combined with the culture medium. Here, a mixture of culture medium and gaseous fluid is pumped out of the chamber top compartment 5a through the outlet 7a. A degassing valve 57 is provided in a flow path between the outlet 7a and the oxygenator 50, to separate gaseous fluid and the liquid culture medium from each other, for efficient oxygenation of the culture medium in the oxygenator 50.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention. However other suitable methods and materials known in the art can also be used. The materials and examples are illustrative only and not intended to be limiting, unless so indicated. For the purpose of clarity and a concise description, features are described herein as part of the same or separate aspects and preferred embodiments thereof, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.In the description above, it will be understood that when an element such as layer, region or substrate is referred to as being “on” or “onto” another element, the element is either directly on the other element, or intervening elements may also be present. Also, it will be understood that the values given in the description above, are given by way of example and that other values may be possible and/or may be strived for.

Furthermore, the invention may also be embodied with fewer components than provided in the embodiments described here, wherein one component carries out multiple functions. Just as well may the invention be embodied using more elements than depicted in the figures, wherein functions carried out by one component in the embodiment provided are distributed over multiple components. It is to be noted that the figures are only a schematic representation of an embodiment of the invention that is given by way of non-limiting example.

For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.

The word ‘comprising’ does not exclude the presence of other features or steps than those listed in a claim. It also does not exclude that nothing else is present, i.e. it may also encompass the meaning of ‘consisting of. Furthermore, the words 'a' and 'an' shall not be construed as limited to 'only one', but instead are used to mean 'at least one', and do not exclude a plurality.

A person skilled in the art will readily appreciate that various parameters and values thereof disclosed in the description may be modified and that various embodiments disclosed and/or claimed may be combined without departing from the scope of the invention.

Claims

1. A cell culture bag for cultivating cells under rocking conditions in a bioreactor system, the bag comprising a deformable flexible wall defining an expandable cell culture chamber for containing the cells in a cell culture medium, and a filter arranged inside the culture chamber dividing the culture chamber in a chamber top compartment for containing cell -free culture medium and a chamber bottom compartment for maintaining the cells, the chamber top compartment provided with an inlet port for filling the cell culture chamber and the chamber bottom compartment provided with an outlet port for draining of the culture chamber, wherein the filter comprises a membrane with a pore size configured to allow exchange of nutrients and gasses between the chamber bottom compartment and chamber top compartment while retaining cells in the chamber bottom compartment, characterized in that the filter is connected to the deformable flexible wall via a filter support frame that is configured and arranged to support the filter and mitigate forces acting on the filter when filling and/or expanding and/or rocking the bag.

2. Cell culture bag according to claim 1, characterized in that the flexible wall is made of a polymer material, and preferably comprises one or more of EVE, EVOH, PE and PVC.

3. Cell culture bag according to claim 1 or claim 2, characterized in that the support frame comprises a substantially rigid body made of the same material as the flexible wall.

4. Cell culture bag according to any one of the foregoing claims, characterized in that the support frame comprises an inner edge section that is attached to the filter and an outer edge section that is attached to the flexible wall.

5. Cell culture bag according to any one of the foregoing claims, characterized in that the filter comprises a stretchable support layer attached to the membrane, which stretchable support layer is configured to allow stretching of the filter.

6. Cell culture bag according to claim 5, characterized in that the stretchable support layer is made of a woven material.

7. Cell culture bag according to any one of claims 5-6, characterized in that the stretchable support layer is attached to the support frame, particularly welded to the support frame.

8. Cell culture bag according to any one of the foregoing claims, characterized in that the filter comprises an antifolding layer attached to the membrane and configured to protect the membrane against folding.

9. Cell culture bag according to claim 7, characterized in that the antifolding layer comprises a rigid mesh structure.

10. A bioreactor system comprising a cell culture bag according any one of the foregoing claims and comprising a fluid conduit circuit that defines a first culture medium flow path that extends from an outlet of the cell culture chamber to the inlet port of the chamber top compartment, and a second culture medium flow path that extends from the outlet of the cell culture chamber to an inlet port of the chamber bottom compartment, and optionally further comprising an agitator unit arranged for operative coupling with the cell culture bag to agitate the cell culture and the fluid cell medium in the cell culture chamber and a mechanism for pinching the outer wall of the variable volume container to create a partially flattened section and operable for increasing the cell-culture chamber in volume by decreasing the flattened section.

11. Bioreactor system according to claim 10, wherein a one-way valve is provided in the second culture medium flow path.

12. Bioreactor system according to claim 10 or 11, comprising an oxygenator arranged in the first culture medium flow path and/or the second culture medium flow path for oxygenating the cell culture medium.

13. Bioreactor system optionally according to any one of claims 10-12, comprising a cell culture bag according to any claims 1-9, and a gas source fluidly connected to the inlet port of the chamber top compartment arranged for supplying a gaseous fluid to the chamber top compartment of the cell culture bag.

14. Bioreactor system according to claim 12 and claim 13, wherein the gas source is provided by the oxygenator.

15. Cell filtration device comprising a filter with a membrane having a pore size configured to allow exchange of nutrients and gasses from one side of the membrane to an opposite side of the membrane by diffusion while retaining cells on one side of the membrane, and comprising a filter support frame to which the filter is connected the support frame configured and arranged to support the filter and mitigate stretching forces acting on the filter.