Bag assembly for cell culture

Abstract

A flexible pouch assembly for cell culture includes one or more pouches forming a plurality of culture compartments wherein a drain port in at least a first culture compartment is adapted to fluidly connect with a second culture compartment when a valve arrangement is open. The invention also discloses a bioreactor for mounting the bag assembly on the rocking tray and a method for culturing cells in the assembly.

Description

[0001] This application is a divisional application of PCT patent application PCT / EP2015 / 059054 (International filing date: 27 April 2015, priority date: 28 May 2014, Chinese national application number: 201580027731.8, invention title: "Bag assembly for cell culture") which entered the Chinese national phase on 25 November 2016. TECHNICAL FIELD

[0002] The present invention relates to bioreactors for cell culture, more specifically to flexible bag bioreactors suitable for multi-stage expansion of cell cultures, such as seed train expansion or cell expansion for cell therapy. The present invention also relates to a method of expanding a cell culture in a flexible bag bioreactor. BACKGROUND

[0003] When a cell culture is expanded from a small cell bank sample to a larger production batch, this usually has to be done in several steps using separate bioreactors. This culture sequence is often referred to as a seed train and is necessary to keep the cell density in a certain optimal window, usually in the range of >10 5 cells per milliliter. In the expansion from a small cryogenic storage vial sample to large scale production of biopharmaceuticals of up to several m 3 These seed trains can involve up to six steps and take several weeks. It is also a complex procedure as the culture has to be transferred aseptically from one bioreactor to another, which has to be done in a LAF workstation or a sterile clean room. There is even a certain risk of accidental contamination under these conditions, which can have catastrophic consequences for a high value large scale culture. Similar problems apply to the expansion of cells to be used in clinical cell therapy, such as stem cells.

[0004] Under the general trend towards single use containers for cell cultures, there is a gradual move towards using flexible bag bioreactors in seed trains. However, there is still a need to empty a smaller bag and transfer the contents to a larger bag, and this is a labor intensive operation with certain contamination risks. It has been proposed to gradually increase the culture volume in a flexible bag by pinching off a section of the bag across its cross section and then removing the pin (WO2008153401), or by starting with a folded bag and then stretching it (US20100055764). However, these solutions do not provide a good seal between the used and unused compartments, leading to leakage of the culture into the unused compartment and contamination of the cell culture with substances released by cells growing under suboptimal conditions. These methods also involve a considerable risk of mechanical damage to the bag, with the risk of bag rupture.

[0005] There is thus a need for a safe and convenient way of transferring cell cultures from one flexible cultivation pod to another in aseptic conditions. There is also a need for precise temperature control of sensitive cell cultures, especially in small volumes. SUMMARY

[0006] One aspect of the present invention is to provide a flexible bag assembly which allows for transfer of cell cultures from one cultivation pod to another without risk of premature leakage between the pods or bags. This is achieved with the assembly defined in claim 1.

[0007] One advantage is that the cultivation pods can conveniently be placed on a single tray and be excited with the same excitation means. A further advantage is that the cultivation can easily be transferred using e.g. gravity or gas pressure.

[0008] A second aspect of the present invention is to provide a bioreactor assembly with flexible bags on a rocking platform which allows for transfer of cell cultures from one cultivation pod to another without risk of premature leakage between the pods or bags. This is achieved with the assembly defined in claim 1.

[0009] A third aspect of the present invention is to provide a cultivation method which allows for safe and convenient transfer of cell cultures from one cultivation pod to another. This is achieved with the method defined in claim 1.

[0010] A fourth aspect of the present invention is to provide a flexible bag assembly which allows for cell cultivation in at least one cultivation pod at essentially constant temperature. This is achieved with the assembly defined in claim 1.

[0011] A fifth aspect of the present invention is to provide a bioreactor assembly which allows for cell cultivation in at least one cultivation pod at essentially constant temperature. This is achieved with the assembly defined in claim 1.

[0012] A sixth aspect of the present invention is to provide a cultivation method in which cells are cultivated at essentially constant temperature. This is achieved with the method defined in claim 1.

[0013] Further suitable embodiments of the present invention are described in the dependent claims. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 An embodiment of the present invention is shown in which three connectable bags are placed side by side on a tray.

[0015] Figure 2 An embodiment of the present invention is shown in which one bag has three connectable cultivation pods.

[0016] Figure 3 Another embodiment of the application is shown, where one bag has three connectable culture compartments.

[0017] Figure 4 An embodiment of the application is shown, where three connectable bags are stacked on a tray.

[0018] Figure 5 The culture sequence in three bags of Figure 4 is shown, starting in the first bag at the bottom of the stack. a) culture in the first bag, b) culture in the second bag and c) culture in the third bag.

[0019] Figure 6 An alternative sequence of Figure 5 is shown, where used bags are removed before culturing in the next bag. a) culture in the first bag, b) culture in the second bag and c) culture in the third bag.

[0020] Figure 7 An alternative stacked bag arrangement is shown, where the first bag is at the top of the stack. a) culture in the first bag, b) culture in the second bag and c) culture in the third bag.

[0021] Figure 8 Two examples of bag-to-bag connections are shown. a) a dip tube drain port in the first bag is connected to an inlet port in the second bag through tubing in a loop above the stack, b) a drain port in the first bag is connected to an inlet port in the second bag through tubing in a loop outside the stack.

[0022] Figure 9 Two examples of drain ports are shown: a) a dip tube for use with bags in a horizontal position, and b) a port for use with bags in an inclined position.

[0023] Figure 10 An embodiment of the application is shown, where one bag has three connectable culture compartments and a temperature control compartment. DETAILED DESCRIPTION

[0024] In one aspect, as Figures 1-10In a bag for cell culture, a flexible bag assembly 1 ; 21 ; 41 ; 61 ; 81 is disclosed. The assembly comprises one or more bags 2, 3, 4; 22; 42; 62, 63, 64, 82, e.g. one or more flexible and / or collapsible bags, forming a plurality of culture compartments 5, 6, 7; 25, 26, 27; 45, 46, 47; 65, 66, 67, 85, 86, 87, e.g. two, three or more compartments. A first drain port 8; 28; 48; 68; 88 in at least a first culture compartment 5; 25; 45; 65; 85 is adapted to be in flow communication with a second culture compartment 6; 26; 46; 66; 86 when a first valve device 10; 30; 50; 70; 90 is opened. The second culture compartment can be larger than the first culture compartment, e.g. having a volume of at least 120%, e.g. at least 150% or 120-1000% of the volume of the first culture compartment. The drain port can be suitably positioned in a low point of the first culture compartment to facilitate complete draining of the compartment. The low point can here mean a point positioned within the lowest 20%, 10% or 5% of the volume of the compartment either when the compartment is in a culture position or when it is moved to a transfer or drain position. In Figure 9 Two examples of drain ports are shown in Figure 9 a) a dip tube 68 is shown which can be used with a bag in a culture position, and Figure 9 b) a flat port 68 is shown positioned at the end of the bag which is adapted to be used for draining with the port in the lower end of the bag when the bag is in an inclined or vertical drain position. As Figure 8a) also a dip tube at the end of the bag can be used as a drain port. This arrangement allows for draining in the horizontal and tilted / vertical positions. The valve arrangement can be in the closed position while the cells are cultivated in the first cultivation compartment, then moved / repositioned in the open position when the cultivation is to be transferred to the second compartment. It can then optionally be moved / repositioned in the closed position to prevent leakage back into the first compartment during cultivation in the second cultivation compartment. Alternatively, the valve arrangement can also comprise a check valve (not shown) that only allows flow in the direction from the first compartment to the second compartment. The valve arrangement can be a valve, such as a pinch valve, a diaphragm valve, a flap valve, a ball valve, a gate valve, a needle valve or a piston valve, but it can also be a clamp, such as a pinch clamp, or it can be a breakable closure, such as a breakable web or a breakable weld between the compartments. The valve arrangement can be reversible, i.e. it can be moved / repositioned from the open position back to the closed position, or it can be non-reversible, i.e. it can only be moved / repositioned from the closed position to the open position. Valves and clamps are examples of reversible valve arrangements and breakable closures of non-reversible valve arrangements. Pinch valves and pinch clamps have advantages from a sterility / contamination point of view, since they can be applied from the outside on the tubing or conduit and do not have any wetted parts. Pinch valves or pinch clamps suitable for application on tubing or conduit up to 10 mm internal diameter or up to 15 mm external diameter are particularly suitable, since they are simple and inexpensive components that provide efficient closing and opening. The function of the valve arrangement is to prevent any leakage of the cultivation in the first compartment into the second compartment when in the closed position, and to allow easy flow from the first compartment to the second compartment when in the open position. If the valve arrangement is reversible, or comprises a check valve, the valve can also have the function of preventing backflow of the cultivation into the first compartment during cultivation in the second compartment, if desired.

[0025] The bags 2, 3, 4; 22; 42; 62, 63, 64, 82 can be made of one or more soft plastic films or sheets that are joined and sealed, e.g. by welding. The films / sheets can comprise polyolefins, such as polyethylene and / or ethylene-vinyl-acetate copolymers, but also barrier layers, such as polyethylene vinyl alcohol polymers and / or tear resistant layers, such as polyamides. The films or sheets can have a thickness of, e.g., 50-300 micrometers, such as 100-250 micrometers.

[0026] The bag assemblies of the present application can conveniently be provided on a rocking tray platform 13; 33; 73; 93 in order to provide agitation during cultivation. The tray can be rocked back and forth around, e.g., an axis 16; 36; 76, 96 placed some distance below the tray. Rocking tray platforms suitable for this purpose are described in, e.g., US 6,190,913, which is incorporated herein by reference in its entirety, and are commercially available from GE Healthcare Bio-Sciences AB as WAVE Bioreactor® and Celsis® Bioreactor, trademarks. TM trademarks.

[0027] In certain embodiments, the flexible bag assembly 1; 21; 41; 61; 81 comprises at least three culture compartments and has a second drain port 9; 29; 49; 89 in the second culture compartment 6; 26; 46; 66, 86 adapted to be in flow communication with the third culture compartment 7; 27; 47; 67; 87 when the second valve means 11; 31; 51, 91 is open. The second drain port and the second valve means can be configured as described above for the first drain port and the first valve means. The third culture compartment can be larger than the second culture compartment, e.g. have at least 120% of the volume of the second culture compartment, e.g. at least 150% or 120-1000% of the volume, and / or at least 140% of the volume of the first culture compartment, e.g. at least 200% or 140-10000% of the volume. This allows for a safe and convenient three-stage expansion from the first culture compartment to the second culture compartment and then to the third culture compartment.

[0028] In Figure 1 and Figure 4 certain embodiments, the flexible bag assembly 1; 61 comprises at least two bags which are suitably connected by a tube 12. The tube can be connected to the drain port 8, 9 in the first bag and to the culture inlet 17 in the second bag. The valve means 10, 11 can be a) valve members connected by the tube, b) valves integrated with the tube or c) externally applied means, e.g. pinch clamps or pinch valves. All these valve means are capable of preventing leakage in the closed position and allowing flow in the open position. The bags can be adapted to be placed side by side with each other on a tray 13 as shown in Figure 1 or they can be adapted to be stacked on a tray 73 with each other as shown in Figures 4-7 It is also possible to have a combination of bags placed side by side with each other and stacked on each other, e.g. two or three bags placed side by side with each other and one larger bag placed underneath or above the two or three bags as shown in Figure 1 The side by side arrangement has the advantage of easy preparation of the assembly and set up on a tray and simple handling. The stacked set up has the advantage of more efficient use of the tray area. When the bags are stacked, it is advantageous to make the connection by a tube and the tube extends in a loop outside or above the stack. This improves accessibility and allows easy handling of the valve means if it is placed in the loop. For example, the bags can be stacked as shown in Figure 5 and Figure 6 where the second bag is on top of the first bag and optionally a third bag (and another bag) is on top of the second bag. This arrangement has the advantage that any sensors on the bottom side of the bags can be kept in contact with contact sensors on the surface of the tray. In particular, the bags can be stacked as shown in Figure 6In the illustrated sequence of culturing, the first bag is removed from the stack after the culture has been transferred to the second bag, and the second bag is optionally removed after the culture has been transferred to the third bag, which allows the sensors in all bags to remain in contact with the same contact sensor on the tray surface. This sequence also has the advantage that the entire bag area is in contact with the tray surface during culturing, which facilitates heat transfer from the heating elements positioned on the tray surface and improves temperature control. Alternatively, the bags can be stacked as illustrated in Figure 7 where the second bag is positioned below the first bag, and optionally the third bag (and another bag) is positioned below the second bag. This arrangement allows easier tightening of the connections, since the drain port of the previous bag can be easily connected to the inlet port on the free top surface of the following bag through the tubing in the loop above the stack. Another advantage is that it is easier to remove the sample in each bag from the sampling port on the free top surface of the bag. If desired, a sheet heating element can be interchanged with the bags in the stack, thereby improving temperature control. For all stack arrangements, if the used bag is removed from the stack after the culture has been transferred to the following bag, this can easily be done by disassembly by, e.g., heat sealing, if the connecting tubing is thermoplastic (e.g., made of soft PVC or a thermoelastic material). Equipment for heat sealing of thermoplastic tubing is readily available, e.g., under the name Hot Lips Tube Sealer (GE Healthcare Bio-Sciences AB).

[0029] In Figures 2-3 and Figure 10 some embodiments, the flexible bag assembly 21; 41 comprises at least one multi-compartment bag 22; 42; 82 having culture compartments 25, 26, 27; 45, 46, 47; 85, 86, 87 connected or connectable by internal conduits 32; 92. The internal conduits can suitably have an internal diameter (circle equivalent) of less than 10%, e.g., less than 5%, of the cross section of the bag. As Figure 2 and Figure 10As shown in the figures, the compartments can be delimited from each other by e.g. walls or welds, which extend at least over a major part of the bag cross section, e.g. over the entire cross section. The inner conduit 32; 92 can in this case be e.g. a hollow part of the ports 28, 29; 88, 89 in the walls, or short pipe sections inserted in the welds as the ports 28, 29; 88, 89. The valve means 30, 31; 90, 91 can be integrated in the conduit, or applied from the outside as clamps or pinch valves. The inner conduit 32; 92 can alternatively be a part of the bag cross section, where the walls or welds do not extend to this part, i.e. the walls / welds only extend over a part of the bag cross section, e.g. over 90-99.9% or 95-99.5% of the bag cross section, leaving the rest of the cross section open, thereby forming the inner conduit 32; 92. The valve means can in this case suitably be clamps applied from the outside, which can provide a leak-tight seal, as they only have to be applied over a short distance.

[0030] In Figure 10 In some embodiments as shown, the flexible bag assembly 81 further comprises at least one bag 82 having at least one temperature control compartment 98. The bag 82 can suitably comprise a top film 99, a bottom film 100 and a delimiting film 97, wherein the delimiting film delimits the temperature control compartment 98 from the culture compartments 85, 86, 87. The temperature control compartment can suitably comprise at least one inlet (not shown) for a thermostating fluid. It can further comprise an outlet (not shown) for the thermostating fluid. The thermostating fluid can be e.g. water or cell culture medium. The thermostating fluid, e.g. water, can be circulated back and forth through the temperature control compartment to a thermostating device, e.g. a thermostating bath, through the inlet and outlet. Alternatively, the temperature control compartment can suitably be positioned in direct contact with heating elements on at least one temperature control surface or tray 93. In this case, the thermostating fluid, e.g. water or medium, does not need to be circulated, but can be used as a temperature buffer. If the temperature control fluid is cell culture medium, the temperature control compartment can be fluidly connected to the first, second or third culture compartment through a drain port and a valve means (not shown), which can then be used as another culture compartment in the same way as discussed above. The advantage of having a temperature control compartment is that the temperature can be more difficult to control accurately enough in small culture compartments by conventional means having a temperature sensor in the culture compartment and controlling the temperature by means of a temperature control (heating) surface on the tray with a feedback loop. With a temperature control compartment, better temperature stability can be obtained due to the heat capacity of the thermostating fluid. The effect of the temperature control compartment is most pronounced for culture compartments having a volume of 1 L or less, e.g. 250 mL or less, 100 mL or less, 50-100 mL, 50-250 mL or 50-100 mL. This applies especially to the first culture compartment. The volume of the temperature control compartment can suitably be at least 1 L, e.g. at least 2 L or at least 5 L or at least 10 times, e.g. at least 20 times the volume of the first culture compartment.

[0031] In Figure 3 In certain embodiments as shown, the flexible bag assembly 41 comprises at least one multi-compartment bag 42 having culture compartments 45, 46, 47 which can be connected by at least one breakable seal 52. The breakable seal can for example be a weak weld as disclosed in EP 2,226,058 Al or US 4,519,499 which are incorporated herein in their entirety by reference. The breakable seal can for example constitute the entire boundary between two adjacent compartments. In this case the weld is broken from the outside of the bag by explosion or other mechanical action, so that the first compartment opens into the second compartment and a second culture step is performed in the connecting compartment. This arrangement has the advantage that no additional components are required. A further advantage is that, although in Figure 3 While separate gas inlets 54 and outlets 55 are shown for each compartment, the culture can be performed with only one gas inlet and one gas outlet for the entire bag with all compartments.

[0032] In certain embodiments of the flexible bag assembly, each culture compartment comprises a gas inlet 14; 34; 54; 74; 94 and a gas outlet 15; 35; 55; 75; 95. These inlets and outlets can be equipped with sterile filters (not shown) to prevent contamination of the culture and for feeding the culture with e.g. air / oxygen and removing gaseous metabolites such as carbon dioxide. They can also be used to feed gas pressure in order to transfer culture liquid from one compartment to another. The gas can for example be fed through the gas inlet while either the gas outlet is closed or simply relies on the outlet sterile filter to provide a sufficiently high back pressure for the liquid transport. The culture compartments can also comprise one or more sampling outlets, inlets for culture medium and sensors for e.g. oxygen or metabolite temperature, cell density, pH and concentration.

[0033] The bag assemblies disclosed above can be suitably supplied, pre-assembled and pre-sterilized, for example by irradiation sterilization, e.g. by gamma or electron beam irradiation. All materials in contact with the liquid are suitably selected to be radiation stable and to impart low levels of extractables after irradiation. All materials can have e.g. USP VI quality.

[0034] In Figures 1-4In a second aspect, the present invention discloses a bioreactor assembly comprising the flexible bag assembly 1 ; 21 ; 41 ; 61 ; 81 disclosed above mounted on a tray 13; 33; 53; 73; 93 adapted to swing back and forth around at least one axis 16; 36; 56; 76; 96. The swinging mechanism and support for the tray are not shown in these figures but are described in detail in US 6,190,913 and V Singh: Cytotechnology 30(1-3), 149-158 (1999). The bioreactor can further comprise a cell culture in at least one of the culture compartments 25, 26, 27; 45, 46, 47; 65, 66, 67; 85, 86, 87. The tray can be equipped with a temperature controlled (heating) surface in direct contact with at least one of the bags. It can further be equipped with sensor connectors in electrical contact with at least one of the bags. At least one of the culture compartments can be connected to a gas supply through a gas inlet and a sterile filter.

[0035] In a third aspect, the present invention discloses a method of culturing cells. The method comprises the steps of: a) providing a bioreactor as disclosed above; b) introducing a culture medium and cells in the first culture compartment 5; 25; 45; 65; 85; c) culturing the cells in the first culture compartment, thereby providing a first cell culture; d) opening the first valve arrangement 10; 30; 50; 70; 90, so as to fluidly connect the first culture compartment with the second culture compartment 6; 26; 46; 66; 86; e) transferring the first cell culture to the second culture compartment; f) introducing a culture medium into the second culture compartment; and g) culturing the cells in the second culture compartment, thereby providing a second cell culture.

[0036] The culturing in the first and second culture compartments can be done using methods well known in the art and described in e.g. V Singh: Cytotechnology 30(1-3), 149-158 (1999) or Clincke et al. Biotechnol. Prog., 2013, Vol. 29, No. 3. Air or other gas can be supplied through the gas inlet 14; 34; 74; 94 and excess air / gas can be expelled through the gas outlet 15; 35; 75; 95 together with gaseous metabolites (e.g. carbon dioxide). The culturing in the first compartment can continue until a predetermined viable cell density (VCD) is reached, e.g. 1.0 x 10 5 , 2.0 x 10 5 , 5.0 x 10 5 or 1.0 x 106 VCD can be measured using e.g. an inline biomass sensor, such as described in US 8,180,575 or WO 2010 / 010313 A2, which are hereby incorporated by reference in their entirety. When the predetermined VCD has been reached, step d) and further steps can be initiated. The transfer in step e) can be done by gravity, e.g. by tilting the tray with the bags or by making it vertically ascending. It can also be done by gas pressure, which is suitably transmitted through the gas inlet of the first bag, in which case the back pressure of the sterile filter on the gas outlet can be sufficient to allow the transfer. The transfer can also be done by other methods, e.g. by applying pressure on the first compartment or by a combination of different methods.

[0037] In certain embodiments, the method further comprises the step e') of closing the first valve means after step e). Alternatively or additionally, the first and second compartments can be disconnected from each other by e.g. disconnecting and sealing any tubing 12; 72 between the compartments. In case the compartments are located in separate bags, this can remove the first bag from the tray, which as discussed above can improve heat transfer and / or allow contact with contact sensors on the surface of the tray.

[0038] In certain embodiments, the method further comprises the steps of: h) opening the second valve means to flow communicatively connect the second cultivation compartment with a third cultivation compartment; i) transferring the second cell culture to the third cultivation compartment; j) introducing culture medium into the third cultivation compartment; and k) cultivating cells in the third cultivation compartment, thereby providing a third cell culture.

[0039] The method can further comprise the step i') of closing the second valve means after step i). Alternatively or additionally, the second and third compartments can be disconnected from each other by e.g. disconnecting and sealing any tubing 12; 72 between the compartments.

[0040] In certain embodiments, step k) and / or g) can be performed in a perfusion mode, i.e. by passing at least part of the culture through a filter, wherein the filtrate is removed and the cells are passed back to the culture and the removed filtrate is replaced with fresh culture medium. This allows further increase of the VCD.

[0041] In a fourth aspect, the present invention discloses a flexible bag assembly for cell cultivation. The assembly comprises at least one bag 82 having at least one cultivation compartment 85, 86, 87 and at least one temperature control compartment 98. The temperature control compartment can be suitably adapted to rest on a tray holder, with the cultivation compartments located above the temperature control compartment. The cultivation compartments can be flow communicatively connected as disclosed above, but they can also be separate and adapted for parallel cultivation. AsFigure 10 As shown, the bag 82 can comprise a top film 99, a bottom film 100 and a delimiting film 97, wherein the delimiting film defines at least one temperature control compartment 98 with at least one culture compartment 85, 86, 87, e.g. the border to each culture compartment. The culture compartment, e.g. each culture compartment, can have a volume of e.g. less than 1 L, e.g. 50-250 mL, and / or the volume of the temperature control compartment can be at least 10 times larger than the volume of the at least one culture compartment, e.g. each culture compartment. The temperature control compartment can comprise e.g. an inlet and an outlet for a temperature regulating fluid, allowing circulation of the temperature regulating fluid from e.g. a temperature regulating bath. Alternatively, the temperature control compartment can have an inlet for a temperature regulating fluid, allowing cultivation and use as a temperature buffer under conditions where the temperature control compartment is closed.

[0042] In a fifth aspect, the present invention discloses a bioreactor assembly comprising: at least one bag 82 or bottle having at least one culture compartment 85, 86, 87; b) at least one flexible temperature control compartment 98 comprising a temperature regulating fluid; and c) a tray 93 or a stack of trays adapted to swing back and forth around at least one axis 96.

[0043] The at least one flexible temperature control compartment is mounted or placed on the tray and the at least one bag or bottle is positioned on top of the at least one flexible temperature control compartment. Thus, heat transfer between the temperature regulating fluid and the cell culture in the culture compartment can be efficiently performed and even for small culture compartments, e.g. 1 L or less, e.g. 250 mL or less, 100 mL or less, 50-100 mL, 50-250 mL or 50-100 mL volume culture compartments, the heat capacity of the temperature regulating fluid provides good temperature control within a narrow temperature range. The bag 82 can comprise e.g. one, two, three, four, five, six, seven, eight, nine or ten culture compartments, which can be adapted for parallel cultivation and / or for sequential cultivation as disclosed above. The flexible temperature control compartment can form a part of the at least one bag 82, i.e. the bag 82 can comprise e.g. Figure 10The at least one bag can further comprise a top membrane 99, a bottom membrane 100 and a delimiting membrane 97, wherein the delimiting membrane defines the border of the at least one temperature control compartment 98 and the at least one cultivation compartment 85, 86, 87. With this arrangement, an integrated bag is obtained, which has one or more cultivation compartments at the top of the bag and a temperature control compartment at the bottom. The temperature control compartment can be equipped with an inlet and an outlet for a temperature regulating fluid, allowing circulation of the fluid, e.g. from a temperature regulating bath, through the temperature control compartment. One or more of the cultivation compartments, e.g. each cultivation compartment, can further be equipped with a temperature sensor, which can be electrically or electromagnetically connected to a control unit, allowing temperature control with a feedback loop. The control unit can further be connected to a temperature regulating bath or other temperature control device in the feedback loop. Additionally or alternatively, the temperature control compartment can comprise a temperature sensor, which can be connected to the control unit. The tray can be equipped with a temperature control (heating) surface in direct contact with the at least one bag. It can further be equipped with a sensor connector in electrical contact with the at least one bag. At least one of the cultivation compartments can be connected to a gas supply through a gas inlet and a sterile filter.

[0044] In a sixth aspect, the present application discloses a method of culturing cells. The method comprises the steps of: a) providing a bioreactor assembly as disclosed above; b) introducing a culture medium and cells into the at least one cultivation compartment 85, 86, 87; and d) culturing the cells in the at least one cultivation compartment.

[0045] In certain embodiments, the temperature in step c) is kept within + / - 2°C, e.g. + / - 1 °C or + / - 0.5°C, of the target temperature. The target temperature can depend on the cell type, and can be e.g. 35-38°C, e.g. 36-37°C, for mammalian cells.

[0046] In certain embodiments, a temperature regulating fluid is circulated through the flexible temperature control compartment. This allows for precise temperature control. Alternatively, the temperature control compartment is in contact with a temperature control (heating) surface on the tray, and the temperature is controlled with a control unit and a feedback loop, using a temperature sensor in / on the temperature control compartment.

[0047] In certain embodiments, the at least one cultivation compartment comprises a temperature sensor, which is electrically or electromagnetically connected to a control unit, and the temperature of the temperature regulating fluid and / or the cultivation compartment is controlled by the control unit with a feedback loop.

[0048] The present application is disclosed herein with reference to the accompanying drawings, which comprise examples illustrating the present application and, along with the description, function as the best mode of practicing the same, and also enable others skilled in the art to practice the application, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the present application is defined by the claims, and can include other examples that occur to those skilled in the art. Such other examples are intended to fall within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims. All patents and patent applications mentioned herein are incorporated by reference in their entirety for the complete disclosure they present, as if each patent or patent application was individually and specifically indicated as incorporated by reference in its entirety.

Claims

1. A flexible multi-compartment bag for culturing cells in a series of culturing stages, the bag comprising: a plurality of culturing compartments of different sizes connected in series, wherein each compartment in the series is individually adapted for use in a corresponding stage of the series of culturing stages; and a breakable seal between each two adjacent compartments in the series, wherein the breakable seal is configured to fluidly connect the two adjacent compartments once opened upon completion of cell culturing in one of the two adjacent compartments, such that the cultured cells can be transferred to a subsequent other compartment in the series for a subsequent stage of cell culturing, wherein the plurality of compartments are provided in a stacked arrangement. The subsequent culturing compartment in the series has a volume that is at least 120% of the volume of the previous culturing compartment in the series.

2. The bag of claim 1, wherein, The subsequent culturing compartment in the series has a volume that is at least 150% of the volume of the previous culturing compartment in the series.

3. The bag of claim 1, wherein, 4. The bag of claim 1, further comprising at least one bag comprising at least one temperature controlled compartment. The temperature controlled compartment comprises a temperature regulating fluid inlet and optionally a temperature regulating fluid outlet.

5. The bag of claim 4, wherein, Each culturing compartment comprises a gas inlet and a gas outlet.

6. The bag of claim 1, wherein, The bag is pre-sterilized prior to cell culturing.

7. The bag of claim 1, wherein, The bag is pre-sterilized by radiation.

8. The bag of claim 1, wherein, 9. A bioreactor assembly comprising the bag of claim 1, the bag being mounted on a tray adapted to swing back and forth about at least one axis.

10. A method of culturing cells, comprising the steps of: a) providing the bioreactor assembly of claim 9; b) introducing culture medium and cells into a first culturing compartment in the series; c) culturing the cells in the first culturing compartment to provide a first cell culture; d) breaking the breakable seal to fluidly connect the first culturing compartment with a subsequent second culturing compartment in the series; e) transferring the first cell culture to the second culturing compartment; f) introducing culture medium to the second culturing compartment; and g) culturing the cells in the second culturing compartment to provide a second cell culture.

11. The method of claim 10, further comprising the step e') of closing the seal between the first culturing compartment and the second culturing compartment after step e).

12. The method of claim 10, further comprising the steps of: h) breaking a second breakable seal to fluidly connect the second culturing compartment with a subsequent third culturing compartment in the series; i) transferring the second cell culture to the third culturing compartment; j) introducing culture medium to the third culturing compartment; and k) culturing the cells in the third culturing compartment to provide a third cell culture.

13. The method of claim 12, further comprising the step i') of closing the seal between the second culturing compartment and the third culturing compartment after step i).

14. A flexible multi-compartment bag for culturing cells in a series of culturing stages, the bag comprising: a plurality of culturing compartments of different sizes connected in series, wherein at least some of the series of connected compartments in the series are individually adapted for use in a stage of the series of culturing stages; and a breakable seal between each two adjacent compartments in the series, wherein the breakable seal is configured to fluidly connect the two adjacent compartments once opened upon completion of cell culturing in one of the two adjacent compartments, such that the cultured cells can be transferred to a subsequent other compartment in the series for a subsequent stage of cell culturing, wherein the plurality of compartments are provided in a stacked arrangement. a breakable seal between each two adjacent ones of the series of vessels in series, the breakable seal configured to fluidly connect two adjacent vessels upon opening at the completion of cell culture in one of the two adjacent vessels, such that the cultured cells can be transferred to a subsequent one of the series for a subsequent stage of cell culture, wherein the plurality of vessels are provided in a stacked arrangement.

15. The bag of claim 14, wherein, The subsequent culture vessel in the series has a volume that is at least 120% of the volume of the previous culture vessel in the series.

16. The bag of claim 15, further comprising at least one bag comprising at least one temperature control vessel.

17. The bag of claim 16, wherein, The temperature control vessel comprises a thermoregulatory fluid inlet and optionally a thermoregulatory fluid outlet.

18. The bag of claim 14, wherein, At least some of the culture vessels comprise a gas inlet and a gas outlet.

19. The bag of claim 14, wherein, The bag is pre-sterilized prior to cell culture.

20. A bioreactor assembly comprising the bag of claim 14, the bag mounted on a tray adapted to be rocked back and forth about at least one axis.

21. A method of culturing cells, comprising the steps of: a) providing the bioreactor assembly of claim 20; b) introducing media and cells into a first culture vessel in the series; c) culturing the cells in the first culture vessel to provide a first cell culture; d) breaking a breakable seal to fluidly connect the first culture vessel with a second, subsequent culture vessel in the series; e) transferring the first cell culture to the second culture vessel; f) introducing media to the second culture vessel; and g) culturing the cells in the second culture vessel to provide a second cell culture.

22. The method of claim 21, further comprising the step of e') closing the seal between the first culture vessel and the second culture vessel after step e).

23. The method of claim 21, further comprising the steps of: h) breaking a second breakable seal to fluidly connect the second culture vessel with a third, subsequent culture vessel in the series; i) transferring the second cell culture to the third culture vessel; j) introducing media to the third culture vessel; and k) culturing the cells in the third culture vessel to provide a third cell culture.

24. The method of claim 23, further comprising the step of i') closing the seal between the second culture vessel and the third culture vessel after step i). ​

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