Cell culture systems and methods

Abstract

The present invention relates to a replaceable suspension cell culture device. In particular, the present invention relates to a replaceable device configured to enable the volume of a cell culture region to be changed by automatically transferring cell cultures between cell culture chambers (1:1A, 1B, 1C) of different volumes. Furthermore, the present invention provides a suspension cell culture system.

Description

[Technical field]

[0001] The present invention relates to a cell culture system. In particular, the present invention relates to a system having a configuration in which the volume of a cell culture region can be changed by transferring a cell culture between cell culture chambers having different volumes. Furthermore, the present invention provides a cell culture method. [Background technology]

[0002] The chemical or pharmaceutical industry is particularly interested in the production of specific cellular products and for that purpose requires a large number of bioreactors and manpower as well as the greatest possible energy and resource optimization in order to ensure the highest quality products.

[0003] To obtain the desired product, it is very important to control the cell culture process so that it is carried out in an appropriate manner and under optimal conditions depending on the various characteristics of the process.

[0004] For desired development and growth, cells in suspension must be cultured in increasing volumes as the culture process progresses.

[0005] A variety of vessels of various sizes have been used traditionally to culture cells contained in a medium, however these traditional means have various drawbacks.

[0006] Some of these drawbacks are: -Poor or non-existent quality control. -It is not possible to monitor the culture status in real time. - There is a risk of contamination due to contact with external media during manual transfer from one container to another of larger volume. - Because the culture progresses quickly, strict time management is required. - If the above mentioned time requirement of volume change at a specific time in suspension cell culture process is not respected, the product quality will be reduced due to the cell stress given to the culture. -An exponential increase in both time and human resources invested in optimizing product quality in compliance with the above requirements.

[0007] Currently, there are various systems that try to systematize the culture process to avoid these drawbacks. Some of these systems incorporate solutions such as culturing the cells in bags, which require constant shaking to keep the cells in suspension. An additional problem is that the starting volume is too large, which requires a series of pretreatments when seeding the cells.

[0008] Additionally, other systems that involve manual passaging or transfer of cells result in poor scalability of the process, thereby compromising final quality control of the product. Summary of the Invention

[0009] The present invention proposes a solution to the above mentioned problems by a cell culture system according to claim 1 and by a method for culturing cells according to claim 14. Preferred embodiments of the invention are defined in the dependent claims.

[0010] A first inventive aspect is a cell culture system comprising: a plurality of cell culture chambers configured to culture cells contained within a medium therein, the plurality of cell culture chambers configured to be in fluid communication with one another via a channel network; a fluid flow control means for circulating a fluid through the channel network; A medium adjustment means; a measuring means configured to monitor the condition of the medium and / or the cells; a controller connected to the fluid flow control means and / or to the regulating means and configured to be in data communication with the measuring means; each of the cell culture chambers includes a plurality of fluid inlets and outlets; all of the plurality of cell culture chambers have different internal volumes; The controller, based on the data measured by the measuring means, to operate the adjusting means, and / or and further configured to operate a fluid flow control means whereby the cells contained in the medium in the first chamber are transferred into a second chamber having an internal volume larger than the internal volume of the first chamber, and so on, as required, into chambers having successively larger internal volumes. The system further comprises a cell retention means configured to retain cells within the cell culture chamber.

[0011] Throughout this specification, fluid is understood to mean a liquid medium and / or cells contained within the liquid medium. Several examples of fluids such as culture medium and / or cells contained within the medium are defined within the present invention. Furthermore, throughout this specification, cell culture is understood to mean cells contained within a liquid medium, preferably cells contained within the medium. Similarly, the medium may be a fresh medium, i.e. a medium rich in nutrients and gases, or a medium deficient in nutrients and gases. This distinction of medium based on the amount of nutrients and gases is made because the initial cells are initially contained in an initial medium (containing the minimum nutrients and gases that can carry out cell culture under optimal conditions), which allows the cell culture to begin until the desired end product (cultured cells) is obtained. In the suspension cell culture process carried out in the present system, the medium can be exchanged with fresh medium up to several times, and this medium can even be adjusted as many times as necessary to restore the gases necessary to continue participating in the growth of the cells. This cell culture process is carried out between the different cell culture chambers contained in the present system.

[0012] The transfer of fluids between the cell culture chambers is controlled by the controller operating on the basis of data received from the measuring means. In other words, such transfer is performed by the action of fluid flow control means for circulating the fluids through the channel network. Such fluid flow control means are also responsible for controlling the passage of the initial medium, i.e. fresh medium as well as starved medium, through the channel network, and for controlling the passage of the initial cells or the cells contained in the medium and already cultured. The fluid flow control means can be operated manually or automatically, depending on the characteristics of the system. Nevertheless, the presence of a controller in the system allows the automatic operation of the fluid flow control means and the regulating means when the system determines that this is necessary.

[0013] A fluid inlet / outlet provided in a cell culture chamber is understood to mean an opening or inlet provided in the chamber through which a fluid can be introduced and / or led out. In a particular embodiment of any of the present systems, each cell culture chamber comprises at least one inlet for medium and / or cell culture and at least one outlet for medium and / or cell culture. In another embodiment of any of the present systems, each cell culture chamber comprises an inlet for medium, an inlet for cell culture or initial cells, an outlet for medium, and an outlet for cell culture or cultured cells.

[0014] The main objective of the present invention is to provide a system suitable for culturing cells contained in a medium in an optimal manner. For this purpose, it is necessary to keep cells that are not adapted to the culture demand at that particular point of the cell culture process confined in an enclosure, container or chamber with specific volumetric characteristics. As a result, the system provides a number of chambers communicating with each other, the internal volume of which increases according to the demand of the culture process. In other words, as the cells are cultured, they require gradually larger volumes so that they can expand and grow, and therefore the cells are transferred sequentially from one chamber to another chamber with a larger internal volume until the cell culture process is terminated. During cell culture, the cells are contained in the medium in the chamber and spread throughout the area allowed by the surface of the bottom of the chamber. In the particular example where the cells are suspended in the medium in the chamber, the effect of gravity tends to cause the cells to be suspended in the medium on the bottom of the cell culture chamber.

[0015] A further object of the system is to provide a means for cell culture to be carried out in an optimal and automated manner with cell transfer between chambers at which point the controller determines the cell transfer based on data monitored by the measurement means, and another object is to prevent cells from exiting the cell culture chambers before the controller determines the cell transfer or before the cells are directly delivered as an end product.

[0016] In certain embodiments, all of the multiple cell culture chambers have different bottom sizes, and the controller is configured to operate the fluid flow control means based on data measured by the measurement means such that cells contained in the medium in the first chamber are transferred into a second chamber whose bottom is larger than the bottom of the first chamber.

[0017] The medium is circulated in each cell culture chamber containing cells for the necessary time before it must be transferred to a chamber with a larger internal volume. This circulation of the medium is understood to mean that the medium is withdrawn from one or more chambers and introduced into one or more chambers in a continuous or periodic manner. This means that advantageously, the depleted medium is withdrawn from the chamber together with the waste products produced by the cells during the culture, and fresh or conditioned medium is introduced into the chamber, which can supply the cells with the gases necessary to continue the culture. In other words, the circulation of the medium contributes to the supply of nutrients and gases to the cells, as well as to the discharge of waste products produced by the cells during the culture.

[0018] The system proposes cell retention means configured to retain the cells inside the cell culture chamber so that the medium flow generated during the circulation of the medium does not entrain the cells and does not lead them out of the chamber earlier than necessary. Advantageously, these cell retention means retain the cells inside the cell culture chamber without the risk of the cells being entrained in the medium flow. The cells are retained inside the cell culture chamber until the controller operates flow control means capable of transferring the cells to a chamber with a larger internal volume than the chamber in which they are located, or until the controller operates flow control means capable of leading them out as the final product (cultured cells).

[0019] In certain embodiments, the fluid flow control means comprises a means for driving a fluid, which can drive medium and / or cell culture out of and into the cell culture chamber in a continuous or periodic manner. More particularly, the means for driving a fluid through the channel network can be a peristaltic or centrifugal pump.

[0020] In certain embodiments, the fluid flow control means comprises a pinch valve that controls the passage of fluid through the channel network.

[0021] The regulation means provided in the system are configured to supply the missing gas (i.e. gas already absorbed by the cells) to the starved medium, thereby advantageously restoring the supply capacity of the cell culture.

[0022] The measurement means is configured to monitor the condition of the medium and / or the cells and is further configured to be in data communication with the controller such that, based on the data received by the controller, the controller determines the demand for the cell culture and operates the fluid flow control means and / or the regulating means.

[0023] The fluid flow control means may be operated manually, i.e., by a user at a time desired, or may be operated automatically, i.e., by a controller, based on data monitored by the measurement means.

[0024] Measurement means, such as sensors, serve to monitor the state of the cell culture or its components during the cell culture process in the system. The system further comprises a controller, which will be responsible for automatically operating the fluid flow control means and the regulating means based on the data transmitted from the sensors. The controller is configured to determine various operations regarding the cell culture based on the measurements from the sensors, such as determining when the medium must be adjusted, when the medium must be discarded and therefore when fresh medium must be introduced or when the cells must be transferred to a chamber of larger dimensions. Based on the measurements of the sensors in the medium, the sensors transmit data to the controller in a continuous manner, which allows the controller to automatically operate both the fluid flow control means and the regulating means. Nevertheless, the cell culture process can advantageously be performed automatically, i.e. without requiring a user to be constantly alert and to intervene with the components of the system.

[0025] Data on the state of the medium measured by the measuring means and transmitted to the controller allows the transfer of cells between the cell culture chambers and allows the number of operations of the regulating means or the number of times to change the circulation of the medium.

[0026] Advantageously, the measurement means monitors the state of the cell culture and / or cells in real time during the cell culture process and further enables the system to operate automatically, i.e. without the need for a user to operate any of the system components or to manage the transfer of the contents of the cell culture chamber.

[0027] To determine whether the medium is still suitable for culturing cells, the measurement means monitors the amount of O2 gas, the amount of CO2 gas, and the pH of the medium, as well as the concentrations of nutrients such as glucose and lactate, biomass, and amounts of other metabolites.

[0028] In certain embodiments, the cell retention means is a filtration means disposed within the cell culture chambers and includes a filtration means configured to filter the medium and further configured to retain cells within each cell culture chamber.

[0029] These filtration means, arranged inside each cell culture chamber, retain the cells inside the chamber while allowing the passage of the culture medium, i.e. do not allow the passage of the cells. The filtration means advantageously retain the cells inside the cell culture chamber without creating the risk of the cells being caught in the flow of culture medium circulating through the fluid inlets and outlets during the culture medium circulation phase.

[0030] In certain embodiments, the filtration means of at least one cell culture chamber comprises: a first compartment adapted to contain a culture medium therein; a second compartment adapted to receive therein cells contained within the culture medium; The filter membrane is configured to allow the passage of medium between the compartments and is configured to retain the cells in the second compartment.

[0031] The two compartments are arranged in a cell culture chamber, separated from each other by a filter membrane, so that during circulation of the medium in the first system, nutrients can diffuse between the fresh medium introduced into the first compartment and the medium contained in the second compartment, thereby bringing the two media into equilibrium, i.e. between the circulating medium and the medium contained in the cell culture chamber, thereby providing the cells with the necessary nutrients and gradually removing waste products produced by the cells, such as lactic acid, by the medium withdrawn from the cell culture chamber, which entrains such waste products. The diffusion of nutrients between the two compartments occurs as a result of the filter membrane circulating the medium filtered through the filter membrane between the two compartments.

[0032] In certain embodiments, each compartment includes at least one fluid inlet / outlet.

[0033] The filter membrane advantageously divides the interior space of the cell culture chamber into two compartments, allowing the cells contained in the medium to be confined in one compartment, while allowing the inflow of fresh medium and the outflow of starved medium in the other compartment. In a particular embodiment, the first compartment is suitable for containing the medium in continuous fluid communication with the interior of the second compartment via the filtration means. In particular, the first compartment comprises an inlet and an outlet for the medium. Advantageously, the inlet and outlet for the medium are separated from the second compartment containing the cells contained in the medium, thereby preventing flows or turbulences generated by the throughflow of the medium into and out of the interior of the chamber from affecting the state of the cells.

[0034] In certain embodiments, the first compartment includes a plurality of walls defining therebetween at least one medium distribution channel, the medium distribution channel being connected along its entire extension to the filter membrane and to at least one fluid inlet / outlet of the first compartment.

[0035] Advantageously, the purpose of the channeled structure defined by the walls forming at least one distribution channel is that the distribution channel covers the entire surface of the membrane. In other words, this arrangement of the walls achieves that the medium introduced into the first compartment circulates along the entire surface of the filtering means (filter membrane). Thus, when fresh medium is introduced into the first compartment through the fluid inlet, this fresh medium will move over the entire extension of the first compartment, thereby ensuring that during such movement it sweeps the entire surface of the filter membrane, thus maximizing the surface for the exchange of nutrients and gases with the second compartment containing the cells contained in the medium. Furthermore, the multiple walls allow for an even distribution of nutrients.

[0036] In certain embodiments, a medium distribution channel defined by the plurality of walls includes a first end connected to a medium inlet and a second end connected to a medium outlet.

[0037] In certain embodiments, the first compartment has a smaller internal volume compared to the internal volume of the second compartment.

[0038] Advantageously, the volume available in the second compartment will be maximized to accommodate the larger volume of cells contained within the medium. Advantageously, the residence time of nutrient-poor medium in the first compartment will also be reduced, thereby accelerating the renewal of the medium as it is replaced or conditioned with new fresh medium.

[0039] In certain embodiments, the filtration means of at least one cell culture chamber comprises at least one filter disposed at at least one fluid inlet and outlet of the cell culture chamber. The filter is configured to allow the passage of the medium and is also configured to retain the cells within the chamber. In certain examples, the filter can be configured to retain the cells and cytokines present in the medium within the chamber. This results in a concentrate present within the chamber, reducing the required concentration of the overall medium.

[0040] Advantageously, the presence of filters at the fluid inlets and outlets of the cell culture chambers allows these filters to be replaced with new filters without entering the cell culture chamber itself.

[0041] In certain examples, the retaining means includes a pump configured to circulate the medium through the chamber at a first height relative to a bottom surface of the chamber at a predetermined rate and flow direction, the first height being greater than a second height at which the cells contained within the chamber are disposed relative to the bottom surface of the chamber, thereby preventing the cells from being entrained prematurely out of the cell culture chamber as the medium circulates through the chamber.

[0042] The speed of medium pumping into the cell culture chamber is predefined according to the size of the bottom of the cell culture chamber. In other words, the flow rate per unit surface at which the medium is introduced into the cell culture chamber is determined according to the size of the bottom of the chamber. The larger the bottom of the chamber, the higher the medium flow rate, i.e., the larger the bottom of the chamber, the higher the flow rate that the cells accommodated in the chamber can tolerate. Furthermore, the medium flow rate will depend on the data measured by the means for sensing the cell culture state, i.e., the medium flow rate can vary according to the demand of the cells to be cultured.

[0043] The culture medium is driven by an adjustable pump into the cell culture chamber so that the culture medium is introduced and circulated into the cell culture chamber with a flow direction or a circulation direction. That is, when the cells are introduced into the cell culture chamber, the cell region contained in the culture medium on the bottom surface of the cell culture chamber is located at a second height above the bottom surface of the cell culture chamber due to the influence of gravity. However, the culture medium is pumped into the cell culture chamber with a flow direction in the chamber located at a first height relative to the bottom surface of the chamber. The first height of the flow direction of the culture medium is higher than the second height at which the cells contained in the cell culture chamber are located. Therefore, the predetermined speed at which the culture medium is driven into the cell culture chamber is such that the culture medium flow occurring in the culture chamber does not entrain the cells and extract them from the chamber. As a result, the speed at which the medium is introduced into the chamber and the location of the cells below the flow direction of the medium prevent the flow direction of the medium from becoming perpendicular to the cells, thereby preventing mixing of the cells and preventing turbulence that may engulf the cells and extract them from the chamber earlier than expected. In certain embodiments, the cells are contained in the medium and arranged in rows within the culture chamber, such that, although the cells are arranged at different second heights, all of the cells are always below the flow direction of the medium circulating through the interior of the cell culture chamber.

[0044] The pump is configured to allow a user to control the flow rate and speed of the medium for pumping according to the needs of the cell culture. In certain embodiments, the pump can be a peristaltic pump, or a centrifugal pump, or a differential pressure pump. In certain embodiments, the pump is configured to be automatically controlled by the controller according to the needs of the cell culture.

[0045] By introducing the medium in a flow or circulation direction at a given rate, two spaces in fluid communication with each other are created in the cell culture chamber: the first space where the cells contained in the medium are in a substantially stationary state and distributed on the bottom surface of the chamber at a second height relative to said bottom surface, and the second space where the circulating medium is located at a first height relative to the bottom surface of the cell culture chamber. Thus, due to the concentration difference between both spaces, a diffusion of nutrients occurs between both spaces. This diffusion of nutrients between the medium circulating through the chamber and the cells contained in the medium is responsible for maintaining the same nutrient concentration between the first and second spaces.

[0046] The cells are retained in the cell culture chamber until the controller operates a flow control means that can transfer the cells to a chamber having a larger internal volume than the chamber in which the cells are located, or until the controller operates a flow control means that can output the cells as the final product (cultured cells).

[0047] The circulation of the medium in the chamber advantageously provides a dispersion of nutrients between the fresh medium introduced into the culture chamber and the medium already contained therein, thereby allowing the equilibration of the two media, i.e. the circulating medium and the medium contained in the cell culture chamber, so that the cells are supplied with the necessary nutrients and the waste products produced by the cells are gradually removed by being entrained in the medium leading out of the cell culture chamber. The diffusion of nutrients in the culture chamber occurs as a result of the circulation of fresh medium at a given speed and in the same upward flow direction with respect to the bottom surface of the cell culture chamber.

[0048] The cells are in a nearly stationary state due to the circulation of the medium at a certain speed and being located below the direction of the medium flow, meaning that the system can advantageously confine the cells contained in the medium within the chamber, while the circulation of the medium allows an inflow of fresh medium and an outflow of starved medium. Furthermore, the speed and direction of the medium flow are such as to prevent currents or turbulences generated by the throughflow of medium into and out of the chamber from affecting the state of the cells.

[0049] In certain embodiments, each chamber includes a plurality of walls defining at least one medium distribution channel therebetween, the medium distribution channel being connected to at least one fluid inlet / outlet.

[0050] Advantageously, multiple walls allow for even distribution of nutrients.

[0051] In certain embodiments, the medium distribution channel includes a first end connected to a medium inlet and a second end connected to a medium outlet.

[0052] In one embodiment, the regulating means comprises a conditioning chamber configured to contain a gas therein, the conditioning chamber being connected to the cell culture chamber via a gas permeable membrane.

[0053] In another particular embodiment, the medium conditioning means comprises: a conditioning chamber connected to the channel network and configured to contain a culture medium therein; and means for injecting at least one gas into the conditioning chamber.

[0054] Advantageously, the means for injecting a gas into the medium supplies said medium with a gas that has already been absorbed by the cells during cell culture.

[0055] In a more specific embodiment, the cell culture chamber is disposed within the conditioning chamber such that the compartment of the cell culture chamber in which the cells contained in the medium are stored includes a gas-permeable (but not liquid-permeable) membrane.

[0056] In another particular embodiment, the adjustment means comprises a gas-permeable, liquid-impermeable silicone tube disposed within the interior of the cell culture chamber in a compartment in which the cells contained in the medium are stored.

[0057] In certain embodiments, the system comprises: a medium reservoir connected to the channel network and configured to be in fluid communication with the interior of the cell culture chamber; and / or an initial cell reservoir connected to the channel network and configured to be in fluid communication with the interior of the cell culture chamber; and / or a retentate reservoir connected to the channel network and configured to receive retentate fluid from the cell culture chamber therein; and / or a final product reservoir connected to the channel network and configured to be in fluid communication with the interior of the cell culture chamber.

[0058] The medium reservoir is a reservoir in which the initial medium in which the cells will be cultured is located, or it can be a new medium, i.e. a medium rich in nutrients and gases that is of interest in the cell culture process for the purpose of replacing the medium containing the cells, and a medium that is already considered to be in a state of shortage and is therefore disposable. Advantageously, this medium reservoir allows to supply fresh medium into the chamber when it is needed in the culture process. Furthermore, this medium reservoir can be understood as a reservoir that can also contain a harvest medium inside, which can be introduced into the final cell culture chamber from which the cells are extracted as the final product contained in the harvest medium, since the cells have already reached their final size, i.e. the cell suspension culture process has already ended.

[0059] The initial cell reservoir is a reservoir in which the cells to be cultured in the medium are located. Advantageously, this initial cell reservoir allows the cells to be cultured to be introduced into any of the cell culture chambers. In particular, in a cell culture process that requires, for example, three cell culture chambers, the cells are generally introduced into the chamber with the smallest internal volume first, and then the cells are gradually expanded into the chambers with the larger internal volumes in succession. In another particular embodiment, if a large number of cells are initially present in the first cell culture chamber, these cells are distributed between the first two cell culture chambers, which allows them to be cultured in an optimal manner, and which prevents an excess of cells per volume accommodated for culture.

[0060] The retentate reservoir is a reservoir intended to relate to residual fluids originating from any cell culture chamber. For example, when a medium is considered to be deficient in nutrients and gases and therefore disposable, it can be removed and collected in the retentate reservoir. Advantageously, the fluid retentate reservoir allows for storing therein any residual fluids that have been discarded from inside any cell culture chamber. In other embodiments, the medium discarded due to lack of nutrients and gases can be of interest to the user, for example, to recover metabolites or products of interest secreted from the cells during the culture. Nevertheless, the retentate reservoir is not only a reservoir for storing disposable products, but also a reservoir in which products of interest are stored.

[0061] The final product reservoir is a reservoir in which the final product, i.e. cells already cultured in a medium, is received at the end of the cell culture process. This final product can be understood as cultured cells contained in a medium or as cultured cells contained in a fresh medium, for example a harvest medium, introduced into the last cell culture chamber. Advantageously, this final product reservoir allows for a quick recovery of already cultured cells without intervention on the cell culture chamber.

[0062] When the measuring means detects that the medium circulating in the cell culture chamber or contained in the cell culture chamber is deficient in a nutrient, e.g., glucose, the measuring means is configured to send a signal to the controller to operate the fluid flow control means. The control means is actuated to remove the nutrient-deficient medium circulating in the cell culture chamber, thereby directing the deficient medium to the retentate reservoir. At the start of the removal of the nutrient-deficient medium, the fluid flow control means is operated to introduce new or fresh medium stored in the medium reservoir into the cell culture chamber where the cells are located, so that the cell culture can continue.

[0063] In certain embodiments, the channel network comprises: a first channel circuit adapted for introducing a medium therein; and a second channel circuit adapted to deliver cells contained within the culture medium.

[0064] In particular, a measurement means is connected to the first channel circuit and a fluid flow control means is connected to both circuits.

[0065] Advantageously, having two independent circuits allows each circuit to be specialized for a different function, resulting in a simpler channel design optimized for the system. In other words, the first circuit handles the distribution and transport of culture medium through the system and its components, while the second circuit handles the transport and distribution of cell culture through the system and its components. The risk of cell damage or loss is also advantageously reduced by using circuits with different functions.

[0066] In certain embodiments, a medium reservoir is connected to a first channel circuit, an initial cell reservoir is connected to a second channel circuit, a retentate reservoir is connected to the first channel circuit, and a final product reservoir is connected to the second channel circuit.

[0067] In certain embodiments, the fluid inlets and outlets of at least one cell culture chamber are connected to one another via a first channel circuit and / or via a second channel circuit.

[0068] In certain embodiments, the system includes a pre-chamber, preferably a cell transfection pre-chamber connected to the first cell culture chamber.

[0069] The cell transfection pre-chamber is suitable for performing necessary cell transfection before starting cell culture and expansion in the cell culture chamber. The transfection pre-chamber is configured to control the cell transfection mechanism and conditions. In certain embodiments, the cell transfection mechanism can be electroporation or gene transfection by viral vectors, among others.

[0070] Carrying out cell transfection prior to expanding the cells for culture advantageously allows the cells to be genetically modified to acquire specific conditions for use after being cultured.

[0071] In certain embodiments, the system includes a balancing means configured to balance the cell culture chamber by driving a motor. In certain embodiments, the motor is manually driven by a user. In another particular embodiment, the motor is configured in data communication with the measurement means, whereby when the balancing means determines based on data monitored by the measurement means that the cell culture chamber needs to be balanced, the measurement means sends a signal to the controller to operate the motor. The balancing means is connected to the controller, and its operation can be preset and can be changed based on data obtained by the measurement means.

[0072] Advantageously, the equilibration means allows the system to equilibrate the cell culture chamber during the cell culture process, contributing to the suspension of the cells in the medium.

[0073] In certain embodiments, the measurement means is connected to the channel network and / or integrated inside the cell culture chamber.

[0074] In certain embodiments, the measuring means comprises a biomass sensor, and / or an oxygen sensor, a pH sensor, and / or a CO2 sensor.

[0075] In certain embodiments, the system is a suspension cell culture system adapted to accommodate cells suspended in a medium within a cell culture chamber.

[0076] In another particular embodiment, the system is a cell culture system with attachment of cells to particulates or surfaces. In the case of attachment to particulates, the retention means, in addition to preventing the cells from being extracted from the interior of the cell culture chamber prematurely, also prevents the particulates from being extracted by the flow of medium.

[0077] The cell culture system of the present invention advantageously allows to carry out cell culture of cells in an optimal manner and with tracking and monitoring of the cell culture process in real time. Thus, the system provides thorough time management of the rate of development of the culture carried out in the system. Furthermore, as a result of the system configuration, a safe and automatic expansion of the cells between the cell culture chambers is carried out, thus preventing human intervention during the cell expansion process, advantageously reducing the risk of contamination of the cell culture by contact with the external medium to zero. Furthermore, the system is configured to carry out the transfer of the cell culture sequentially, such as by issuing instructions to transfer the cells to a chamber having a larger internal volume than the chamber in which the cells are initially located, thereby preventing the cell culture from reaching suboptimal nutritional and oxygen conditions. Before the cell culture reaches a suboptimal situation due to the volume in which the cells are located being too small for the culture to proceed, the cell culture is expanded into another chamber having a larger volume that covers what the cells need to continue growing. The transfer or circulation of the cell culture in the system is operated by a control means provided in the system.

[0078] A second inventive aspect provides a method for culturing cells, implemented in a cell culture system according to the first inventive aspect, comprising: a) introducing cells contained in a medium into a first cell culture chamber; b) circulating the medium through the channel network; c) transferring the cells contained in the medium into a second cell culture chamber; d) repeating step b) within a second cell culture chamber; and e) obtaining the cultured cells; The method further comprises monitoring the state of the cell culture and / or the cells by means of measuring means in order to determine when the cells have acquired a particular property and, if necessary, when they have to be transferred sequentially into a chamber with a larger internal volume, such as when they have to be transferred according to step c) into a cell culture chamber with a larger internal volume compared to the cell culture chamber in which they are located.

[0079] The cell culture method of the invention advantageously allows the cell culture of cells contained in a medium to be carried out in an optimal manner and in real time. This method is implemented in the above system providing a thorough time control regarding the rate of development of the culture being carried out. This method achieves a safe and automatic expansion of cells between cell culture chambers, thus preventing human intervention during the cell expansion process, advantageously reducing the risk of contamination of the cell culture by contact with the external medium to zero. Before the cell culture is stressed due to the volume in which the cells contained in the first cell culture chamber are located being too small for the culture to proceed, this cell culture is expanded in another chamber with a larger volume.

[0080] Advantageously, monitoring of the cell culture allows the method to be completed automatically without the need for a user to be present to control the components of the system, and allows the user to be aware of the condition of the medium and / or cells in real time.

[0081] In certain embodiments, step d) of repeating step b) in the second cell culture chamber can be repeated as many times as necessary to reach the target number of cultured cells.

[0082] In certain embodiments, the method further comprises adjusting the medium by the adjusting means if the measuring means determines that the medium is in a deficiency state, preferably a deficiency state in which the medium is deficient in gas and / or nutrients.

[0083] In certain embodiments, the method comprises: This involves extracting the nutrient depleted medium from within the cell culture chamber and introducing new or fresh medium into the cell culture chamber.

[0084] In certain embodiments, the method further comprises equilibrating the cell culture chamber with an equilibration means.

[0085] All features and / or steps of the methods described in this specification (including the claims, description, and drawings) may be combined in any combination, except for combinations of mutually exclusive features.

[0086] The above and other features and advantages of the present invention will be more clearly understood from the following detailed description of preferred embodiments, given by way of illustrative and non-limiting example only with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0087] [Figure 1]FIG. 1 shows a schematic diagram of a cell culture system according to a particular embodiment of the first inventive aspect of the present invention. [Diagram 2] FIG. 2 shows a schematic diagram of a cell culture system illustrating a first channel circuit according to a particular embodiment of the first inventive aspect of the present invention. [Diagram 3] FIG. 3 shows a schematic diagram of a cell culture system illustrating a second channel circuit according to a particular embodiment of the first inventive aspect of the present invention. [Figure 4] FIG. 4 shows a schematic perspective view of a cell culture chamber according to a particular embodiment of the first inventive aspect of the present invention. [Diagram 5] FIG. 5 shows a schematic plan view of the cell culture chamber depicted in FIG. [Figure 6] FIG. 6 shows a schematic partial cross-sectional view of the cell culture chamber depicted in FIG. [Figure 7] FIG. 7 shows a cell culture system according to a particular embodiment of the first inventive aspect of the present invention. [Figure 8] FIG. 8 shows a cell culture system according to a particular embodiment of the first inventive aspect of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0088] Cell Culture Systems Figure 1 shows a specific example of a suspension cell culture system according to a first inventive aspect of the present invention. The system comprises three cell culture chambers (1:1A, 1B, 1C) arranged so as to form a stack on top of each other. Each of these chambers (1A-1C) has an internal volume different from the other chambers, and in particular it can be seen that the internal volume increases gradually from the first chamber (1A) to the second chamber (1B) and further to the third chamber (1C).

[0089] Each of the cell culture chambers (1:1A, 1B, 1C) shown in Fig. 1 includes a plurality of fluid inlets (10.1A-10.4A, 10.1B-10.4B, 10.1C-10.4C) and a filter means (2) as a cell retention means. Each of the chambers (1A-1C) further includes a filter membrane (12) disposed penetrating the interior of the cell culture chamber (1A-1C) such that the interior space of each of the chambers (1A-1C) is divided into a first compartment (14A-14C) and a second compartment (15A-15C), and the first compartment (14A-14C) is disposed above the second compartment (15A-15C) in the normal operating position. The first compartment (14A-14C) includes a first medium inlet (10.1A-10.1C) and a first medium outlet (10.2A-10.2C), both of which are connected to a channel network common to the entire system. The second compartment (15A-15C) includes a cell culture inlet (10.3A-10.3C) and a cell culture outlet (10.4A-10.4C), both of which are connected to a channel network common to the entire system. The illustrated channel network in the system is formed by a first channel circuit (16) and a second channel circuit (17), both of which are connected to each other through the interior of each cell culture chamber (1). The first channel circuit (16) is suitable for delivering a culture medium, while the second channel circuit (17) is suitable for delivering cells suspended in the culture medium.

[0090] The system shown in Figure 1 further comprises flow control means (3:3.1-3.9) for the fluids, which in this particular example are pinch valves, which serve to regulate the passage of fluids through the channel network and to allow the introduction and / or withdrawal of fluids from the cell culture chambers (1A-1C).

[0091] The fluid flow control means comprises a means (4) for driving a fluid through the channel network. In this particular example, the means (4) for driving a fluid is a peristaltic pump (4.1) which serves to draw the fluid from the interior of each cell culture chamber (1A-1C) by pumping and to pump the fluid between the chambers or within the same cell culture chamber (1A-1C) in a uniform manner so as to circulate the fluid throughout the channel network. In particular, Figure 1 illustrates a first pump (4.1) which serves to pump the culture medium and a second pump (4.2) which serves to pump the cells suspended in the culture medium.

[0092] The system shown in Figure 1 further comprises a medium reservoir (7) suitable for containing initial or fresh medium therein, the medium reservoir (7) being connected to a channel network, which allows fresh or new medium to be supplied to the interior of any of the cell culture chambers (1A-1C) as required.

[0093] The system further comprises an initial cell reservoir (8) suitable for containing initial cells to be cultured in suspension inside the cell culture chambers (1A-1C), said initial cell reservoir (8) being connected to a channel network, by which the interior of the cell culture chambers (1A-1C) can be supplied with cells already contained in an initial liquid medium.

[0094] 1 further shows a retentate reservoir (9) suitable for containing retentate fluids leading from any of the cell culture chambers (1A-1C) and connected to a channel network allowing the introduction into the retentate reservoir (9) of nutrient- and gas-poor medium determined to be disposable by the fungus during the suspension cell culture process.

[0095] Figure 1 also shows a final product reservoir (11) connected to the channel network, which is a container that contains already cultured cells that have been removed from any of the cell culture chambers (1A-1C) after a suspension cell culture process.

[0096] FIG. 1 further shows a controller (24) connected to the fluid flow control means (3:3.1-3.9), the regulating means (6), the measuring means (5) and the peristaltic pumps (4.1, 4.2).

[0097] The system of Fig. 1 further comprises a measuring means (5), which in one embodiment is a sensor connected to the channel network. The measuring means (5) serves to measure and monitor the state of the medium and / or the cells, thereby making it possible to know the cell culture state, i.e. the state of the cells in the different suspension cell culture phases. In a particular embodiment, the measuring means (5) is a glucose and lactate sensor connected to the channel network, in particular to the first circuit (16) of the channel network in Fig. 1. The measuring means (5) is connected to a controller (24), so that all data measured and monitored by the measuring means (5) are transmitted to the controller (24), which can take said data into account in operating the fluid flow control means (3:3.1-3.9) and the medium conditioning means (6).

[0098] The system comprises a medium conditioning means (6) connected to the channel network. If the controller (24) determines based on the data measured by the measuring means (5) that the medium is no longer fresh, i.e. that there is a lack of gas for the cell culture, this medium is conditioned by the conditioning means (6) so as to again supply the medium with the gas necessary for continuing the cell culture of the cells suspended in the cell culture chambers (1A-1C). The conditioning means (6) comprises a conditioning chamber (13) into which a medium lacking gas can be introduced and such medium can be conditioned to restore the gas necessary for the cell culture. For the addition of the gas-deficient medium led into the conditioning chamber (13), the system comprises a gas injection means (20).

[0099] On the other hand, if the controller (24) determines that the medium is stale, i.e., lacking nutrients, the fluid flow control means (3:3.1-3.9) operates to discard the medium and direct it to the retentate reservoir (11). New, fresh medium is then introduced into the cell culture chamber (1) in which the cells are located and allowed to continue growing.

[0100] Figure 2 shows a specific example of a cell culture system according to a first inventive aspect of the present invention. The system comprises two cell culture chambers (1A-1B) stacked one on top of the other, with the first chamber (1A) located at the top of the stack having a smaller internal volume compared to the second cell culture chamber (1B) located below. Each cell culture chamber (1A-1B) comprises a medium inlet (10.1A-10.1B) and a medium outlet (10.2A-10.2B), respectively.

[0101] In the system of FIG. 2, the conditioning means (6) comprises two conditioning chambers (13), one for each cell culture chamber (1A-1B). Each conditioning chamber (13) is in contact with the interior of each cell culture chamber (1A-1B) via a gas-permeable membrane (22). The conditioning means (6) further comprises a gas injecting means (20) configured to inject gas into the conditioning chamber (13) and a gas outlet or exhaust means (23) configured to release the gas present in the conditioning chamber (13). In particular, FIG. 2 illustrates only the first channel circuit (16) and its connections to the various components of the system. The first medium inlet (10.1A) and the first medium outlet (10.2A) of the first cell culture chamber (1A) are connected to each other and to the other cell culture chamber (1B) via the first channel circuit (16). Also connected to this first channel circuit (16) are a number of fluid flow control means (3: 3.1-3.9), a means for driving a fluid (4) and a measuring means (5). FIG. 2 further shows a medium reservoir (7) connected to the first channel circuit (16) of the channel network of the system, and a retentate reservoir (9) also connected to the first channel circuit (16). The withdrawal of the medium from the interior of the chambers (1A-1C) via the first medium outlets (10.2A-10.2B) and the subsequent circulation through the first channel circuit (16) are driven by a first pump (4.1) and controlled by the fluid flow control means (3: 3.1-3.9). The introduction of the medium into the interior of the chambers (1A-1C) via the first medium inlets (10.1A-10.1B) is then also controlled by the fluid flow control means (3: 3.1-3.9).

[0102] The system further comprises a controller (not shown) connected to the fluid flow control means (3:3.1-3.9) and the regulating means (6) and in data communication with the measuring means (5). The conditioning of the medium contained and circulating inside the cell culture chambers (1A-1B) is performed by interaction of a nutrient-rich gas injected into the conditioning chamber (13) with the medium via a permeable membrane (22).

[0103] The system also includes a first pump (4.1) (means for driving a fluid) connected to the first channel circuit (16) and configured to circulate the culture medium through the chambers (1A-1B) at a predetermined speed and in a particular flow direction (not shown).

[0104] FIG. 3 shows a specific example of a cell culture system according to the system of FIG. 2. The system comprises two cell culture chambers (1A-1B) stacked one above the other, similar to FIG. 2. Each of the cell culture chambers (1A-1B) comprises a second cell culture inlet (10.3A-10.3B) and a second cell culture outlet (10.4A-10.4B). The system further comprises a regulation means (6) similar to that illustrated in the system of FIG. 2. In particular, FIG. 3 illustrates only the second channel circuit (17) and its connections to the various components of the system. The second cell culture inlets (10.3A-10.3B) and the second cell culture outlets (10.4A-10.4B) of the chambers (1A-1B) are connected to each other via the second channel circuit (17). Also connected to this second channel circuit (17) are a number of fluid flow control means (3: 3.1-3.9) and means for driving the fluids, in particular a second pump (4.2) similar to the first pump (4.1) described in FIG. 2. FIG. 3 further illustrates an initial cell reservoir (8) connected to the second channel circuit (17) of the channel network of the system, and an end product reservoir (11) also connected to the second channel circuit (17). The extraction of the cell culture from the interior of the cell culture chamber through a second cell culture outlet (10.4A-10.4B) and the subsequent circulation of the cell culture through this second channel circuit (17) are driven by the second pump (4.2) and controlled by the fluid flow control means (3: 3.1-3.9). Next, the introduction of cell culture into any of the cell culture chambers (1A-1B) through a second cell culture inlet (10.3A-10.3B) is also controlled by the fluid flow control means (3:3.1-3.9).

[0105] The first channel circuit (16) illustrated in FIG. 2 is suitable for circulating culture medium or harvest medium therethrough, while the second channel circuit (17) illustrated in FIG. 3 is suitable for circulating a cell culture, i.e., cells suspended in culture medium or harvest medium, therethrough.

[0106] Figure 4 shows a perspective view of a particular example of a cell culture chamber (1). The cell culture chamber (1) comprises two internal compartments, namely a second compartment (15) and a first compartment (14) arranged above the second compartment (15). The cell culture chamber (1) further comprises a number of connectors (19) coupled to each of the ports (10.1-10.4) contained within the cell culture chamber (1). These connectors (19) serve to connect each of the ports (10.1-10.4) to the channels / tubes (not shown in the drawings) of the channel network.

[0107] In particular, the cell culture chamber (1) of FIG. 4 shows a first medium inlet (10.1) and a first medium outlet (10.2), both of which are connected to a connector (19) and located at the top of the first compartment (14) of the cell culture chamber (1).

[0108] Furthermore, the cell culture chamber (1) illustrates a second cell culture inlet or primary cell inlet (10.3) and a second cell culture outlet (10.4), both of which are connected to a connector (19) and located in a lateral portion of the second compartment (15) of the cell culture chamber (1). Both compartments (14, 15) are separated from each other by a filter membrane (shown in FIG. 6), which allows fluid communication between both compartments (14, 15) inside the cell culture chamber (1).

[0109] Figure 5 shows a plan view of the interior of the cell culture chamber (1) depicted in Figure 4, with the internal structure of the first compartment (14) and the first medium inlet (10.1) and first medium outlet (10.2) being illustrated in particular by dashed lines. In this particular example, the first compartment (14) comprises a number of walls (14.1) arranged in a circular spiral form that defines a medium distribution channel (18). This medium distribution channel (18) is connected along its entire extension to the filter membrane (12), as depicted in Figure 6. Furthermore, the distribution channel (18) comprises a first end (18.1) connected to the first medium inlet (10.1) and a second end (18.2) connected to the first medium outlet (10.2).

[0110] Thus, when culture medium is introduced into the first compartment (14) through the culture medium inlet (10.1), it flows along the distribution channel (18), sweeping across the entire surface of the filter membrane (12), until it reaches the first culture medium outlet (10.2) through which it is discharged.

[0111] Figure 6 shows a side cross-sectional view of the cell culture chamber (1) depicted in Figures 5 and 6. Figure 6 details the difference in volume between the compartments (14, 15). The first compartment (14) has a smaller internal volume compared to the internal volume of the second compartment (15).

[0112] Fig. 7 shows a particular example of a system according to a first inventive aspect, comprising two cell culture chambers (1:1A, 1B), the first chamber (1A) having a smaller internal volume compared to the internal volume of the second chamber (1B). Each chamber (1A-1B) comprises as a retention means a filter membrane (12) arranged penetrating the interior of the cell culture chamber (1), thereby dividing the internal space of this chamber (1A-1B) into a first compartment (14A-14B) and a second compartment (15A-15B), with the first compartment (14A-14B) being arranged above the second compartment (15A-15B) in the normal operating position. Both cell culture chambers (1A-1B) are connected to each other via a channel network. The circulating medium (26) will be present in the first compartment (14A-14B), but the cells (25) contained in the medium (26) that is filtered between the compartments will be present in the second compartment (15A-15B).

[0113] The system shown in Figure 7 further includes a plurality of valves (3:3.1-3.10) connected to the channel network. The system further includes a pump (4) also connected to the channel network.

[0114] The system depicted in FIG. 7 includes a medium reservoir (7), an initial cell reservoir (8), a retentate reservoir (9), and a final product reservoir (11), all of which are similar to those depicted in FIG.

[0115] The system of FIG. 7 further includes a first sensor (5.1) and a second sensor (5.2), both of which are connected to the channel network and configured to monitor the condition of the culture medium circulating through respective ones of the first cell culture chamber (1A) and the second cell culture chamber (1B).

[0116] The first sensor (5.1) and the second sensor (5.2) in the system shown in Fig. 7 each include a gas sensor and a nutrient sensor, i.e. sensors configured to measure the pH, the level of O2, the level of glucose and the level of lactate contained in the culture medium, respectively. The sensors (5.1, 5.2) are connected to a controller (not shown in the drawings) in such a way that all data measured and monitored by the sensors (5.1, 5.2) are transmitted to the controller in order to operate the fluid flow control means (3:3.1-3.10) and the medium conditioning means (6) according to the needs of the cell culture.

[0117] In the system shown in FIG. 7, the medium conditioning means (6) comprises a conditioning chamber (13) in which gas-deficient medium can be introduced and conditioned, thereby restoring the gas required for cell culture, or the conditioning chamber can also be filled with new fresh medium before pumping towards the cell culture chambers (1A-1B). To condition the gas-deficient medium accumulated in the conditioning chamber (13), the system comprises a gas injection means (20). Furthermore, the system comprises a gas release means (21) for the case of excess pressure occurring in the suspension cell culture process. The gas injection means (20) and the gas release means (21) are also connected to a controller (not shown) and can thus be operated at times determined by the controller.

[0118] FIG. 8 shows a particular example of a system comprising two cell culture chambers (1A, 1B), the first chamber (1A) having a smaller internal volume compared to the internal volume of the second chamber (1B). Both cell culture chambers (1A-1B) are connected to each other via a channel network. The system comprises a peristaltic pump (4) connected to the channel network and serving to drive a culture medium (26) into the interior of the cell culture chambers (1A-1B). The culture medium (26) is driven by such pump (4) at a given speed and flow direction at a first height (h1) relative to the bottom surface of the chambers (1A-1B), which is higher compared to a second height (h2) at which the cells (25) contained inside the chambers (1A-1B) are located relative to the bottom surface of the chambers (1A-1B). Therefore, when the medium circulates through the interior of the chambers (1A to 1B), the cells (25) are prevented from being drawn out of the chambers (1A to 1B) more quickly than necessary.

[0119] In a particular example, the flow direction of the medium circulating through the interior of the chambers (1A to 1C) is substantially parallel to the arrangement of the cells (25) housed within the chambers.

[0120] The system depicted in FIG. 8 further includes a plurality of valves (3:3.1-3.10) connected to the channel network.

[0121] The system depicted in FIG. 8 further includes a medium reservoir (7), an initial cell reservoir (8), a retentate reservoir (9), and a final product reservoir (11), all of which are similar to those depicted in FIG.

[0122] The system of Figure 8 further comprises a first sensor (5.1) and a second sensor (5.2), both connected to the channel network and configured to monitor the condition of the medium circulating through the respective first cell culture chamber (1A) and second cell culture chamber (1B). The sensors (5.1, 5.2) depicted in Figure 8 are identical to those described with respect to Figure 7.

[0123] Furthermore, the system illustrated in FIG. 8 includes conditioning means (6) for the culture medium (26), which are similar to those described with reference to FIG.

[0124] 8, it can be seen that the flow direction (27) of the culture medium (26) circulating through the interior of the chambers (1A, 1B) is located at a first height (h1) relative to the bottom surface of the cell culture chambers (1A, 1B) and that the arrangement of cells (25) contained in the culture medium (26) is located at a second height (h2) relative to the bottom surface of the cell culture chambers (1A, 1B). The first height (h1) is made higher compared to the second height (h2).

[0125] Cell culture method The following describes the various steps of a cell culture method according to the invention, which is implemented in a system as illustrated in Figure 7, which corresponds to the system in the first inventive aspect. In particular, the method is a suspension cell culture method. The method comprises the following steps according to the invention: a) introducing cells (25) suspended in culture medium (26) into a first cell culture chamber (1A). b) circulating the culture medium (26) through the channel network. c) transferring the cells (25) suspended in culture medium (26) into a second cell culture chamber (1B). d) Repeating step b) inside the second cell culture chamber (1B). e) Obtaining the cultured cells (25). Here, the method further comprises monitoring the state of the cell culture and / or the cells (25) by means of measuring means (5) in order to determine when the cells (25) have acquired a particular property and when they must be transferred according to step c) to a cell culture chamber (1B) having a larger internal volume compared to the cell culture chamber in which they are located.

[0126] Before starting the suspension cell culture method, the initial functional state of the system is that all valves (3: 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 3.10, 3.11, 3.12, 3.11) are closed and the pump (4) is stopped.

[0127] Prior to step a), the conditioning chamber (13) is filled with culture medium (26) coming from the culture medium reservoir (7) by opening valves (3.5) and (3.10), which releases pressure from the channel network and allows the culture medium (26) to circulate into the conditioning chamber (13).

[0128] The functional scheme of the system in step a) is as follows: - Operate the pump (4). -Open valve (3.1A). -Open the valve (3.6). -Open valve (3.3A).

[0129] After the cells (25) are suspended in the culture medium (26) inside the first chamber (1A), the culture medium circulates between both compartments (14, 15) of the chamber through the filter membrane (12). The cell culture is introduced into the first chamber (1A) until the culture medium reaches the first sensor (5.1). In the particular example according to the cell culture system illustrated in Figures 2 and 3, the cells are introduced into the first chamber (1A) through the inlet (10.3) together with the culture medium introduced through the inlet (10.1).

[0130] In a particular example, before step b), the cell culture chambers (1A-1B) are equilibrated by a motor (not shown in the drawings) provided in the system. The functional scheme of the system during equilibration is that all valves (3: 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 3.10, 3.11, 3.12, 3.11) are closed and the pump (4) is stopped. In a particular embodiment, the motor provides equilibration of the cell culture chambers (1A-1B) from -10 degrees to 10 degrees.

[0131] The functional scheme of the system in step b) is as follows: -Open valve (3.1A). -Open valve (3.2A). -Open the valve (3.7). - Operate the pump (4).

[0132] In certain embodiments, during the circulation step, the medium (26) is circulated through the channel network and introduced into the first cell culture chamber (1A) and discharged from the first cell culture chamber (1A) after about 30 seconds.

[0133] In step b), in addition to circulating the medium (26) already introduced into the chamber itself, the medium (26) contained in the conditioning chamber (13) is also circulated in combination with other media. It may be necessary to introduce a larger amount of medium (26) for the circulation, by again introducing fresh medium (26) from the fresh medium reservoir (7) into the conditioning chamber (13) as described above.

[0134] In the particular example of the cell culture system illustrated in Figures 2 and 3, after the cells are introduced into the first chamber (1A), the medium is circulated by being introduced through the inlet (10.1) of the cell culture chamber and withdrawn through the outlet (10.2) at a predetermined rate, thereby preventing the cells suspended in chamber (1A) from becoming entrained in the flow of medium through the outlet (10.2).

[0135] If, during step b), the data measured by the first sensor (5.1) and received by the controller (not shown in FIG. 7) determine a lack of gas in the culture medium (26), the controller sends a signal to the operating means (6) and activates the gas injection means (20) by opening the valve (3.11) to allow storage of the culture medium (26) circulating in the conditioning chamber (13), thereby introducing gas into the culture medium (26) for conditioning, which is then introduced back into the chambers (1A-1B) for the purpose of proceeding with the cultivation of the cells.

[0136] If, during step b), the first sensor (5.1) determines that the medium (26) is deficient in nutrients, this first sensor (5.1) outputs a signal to the controller in order to operate some valves so that the nutrient-deficient medium (26) can be discarded, i.e. to direct such medium to the retentate reservoir (9). In this discard phase with respect to the nutrient-deficient medium, the functional scheme of the system is as follows: -Open valve (3.2A). -Open the valve (3.8). -Closing the valve (3.7).

[0137] When the nutrient-deficient medium is discarded, the system supplies fresh medium to the interior of the culture chambers (1A-1B) as described above to continue the cell culture process. After the fresh medium (26) is introduced into the first cell culture chamber (1A) through the channel network, the medium is circulated until the first sensor (5.1) detects that there is a gas or nutrient deficiency in the medium or that the cells must be transferred to the second cell culture chamber (1B). If the controller determines, based on the data measured by the first sensor (5.1), that there is a gas or nutrient deficiency in the medium at a time before the cells (25) must be transferred to the new chamber, the system responds as described above for each of these situations, adjusting the medium (26) circulating through the system or discarding the medium (26) and introducing fresh medium.

[0138] If during step b), the first sensor (5.1) detects that the cells (25) need to be expanded to a larger volume, the controller operates the system to proceed to step c) of transferring the cells (25) suspended in the medium (26) to the second cell culture chamber (1B).

[0139] In step c), the functional scheme of the system is as follows: -Open valve (3.1A). -Open valve (3.3A). -Open the valve (3.3B). - Operate the pump (4).

[0140] In a particular embodiment, in step c) of transferring cells between the cell culture chambers (1A-1B), the equilibration motors are operated (with all valves closed) and these motors equilibrate the cell culture chambers (1A-1B) from -10° to 10°. In a particular example, after equilibration, the equilibration motors are stopped and the cell culture chambers (1A-1B) are left equilibrated at -20° relative to the initial position of the system to allow the cells (25) suspended in the medium (26) to enter the second cell culture chamber (1B).

[0141] After the cells (25) have been transferred to the second cell culture chamber (1B), the culture process continues as described above for the first cell culture chamber (1A), as specified in step d) for circulating the culture medium. During step d), the second sensor (5.2) monitors the condition of the culture medium and the measured data is transmitted to the controller, which determines the condition of the cells (25). If the controller nevertheless determines that there is a gas or nutrient deficiency in the culture medium (26), the controller will operate the system appropriately, as described above for the first cell culture chamber (1A).

[0142] If the second sensor (5.2) detects that the system has already reached the final product, i.e. that the cells (25) have already been cultured, these cells (25) are harvested in step e) of the method.

[0143] In step e), the functional scheme of the system is as follows: -Open the valve (3.3B). -Open the valve (3.9). - Open the valve (3.11). - Stop the pump (4).

[0144] Air is injected into the channel network to assist in the evacuation of the cultured cells suspended in the medium to the final product reservoir (11) by gas injection means (20), which, when valve (3.11) is opened, injects air into the second compartment (15) of the second cell culture chamber (1B) to evacue the final product.

[0145] In a particular embodiment, in step e) of obtaining the final product, the equilibration motors are operated (with all valves closed) and these motors equilibrate the second cell culture chamber (1B) from -10 degrees to 10 degrees. In a particular example of step e), after equilibration, the equilibration motors are stopped and the second cell culture chamber (1B) is equilibrated at -20 degrees with respect to the initial position of the system, so that the final product can be fully delivered to the final product reservoir (11). After collection of the final product, the equilibration motors place the cell culture chamber (1) in the initial position of the system.

[0146] In a particular embodiment, the measuring means (5) comprises a third sensor (not shown in the drawings) connected to the channel network and in data communication with the controller, the third sensor being configured to detect when only air starts to circulate through the channel network, i.e. when the entire final product has already been collected into the final product reservoir (9). The third sensor is located in front of the final product reservoir (11).

[0147] With regard to the suspension cell culture method carried out in the system illustrated in FIG. 8, the same steps are reproduced as those described with respect to the method implemented by the system of FIG. 7. However, in this case, in step b), the circulating medium (26) is driven by the pump (4) at a predetermined speed and flow direction (27) into the chambers (1A-1B) at a first height (h1) relative to the bottom surface of the chambers (1A-1B), which is higher compared to a second height (h2) at which the cells (25) contained in the medium (26) are located, in order to prevent these cells (25) contained in the chambers from being drawn out of the cell culture chambers (1A-1B) too early. In other words, as an alternative to the solution proposed with respect to the system of FIG. 7, in which a filter membrane is combined with the details of the system itself, the system illustrated in FIG. 8 proposes to include not only the cell culture but also the medium with a speed and a predetermined flow direction depending on the characteristics of the system itself. In contrast, therefore, what has been described above with respect to the suspension culture method implemented by the system depicted in FIG. 7 also applies to the suspension culture method implemented by the system of FIG.

Claims

1. 1. A cell culture system comprising: a plurality of cell culture chambers (1A-1C) configured to culture cells contained in a medium therein, the plurality of cell culture chambers (1A-1C) being configured to be in fluid communication with one another via a plurality of channels; a fluid flow control means (3:3.1-3.9) for circulating a fluid through said plurality of channels; A medium adjustment means (6); measuring means (5) configured to monitor the state of the medium and / or the cells; a controller (24) connected to said fluid flow control means (3: 3.1-3.9) and / or to said regulating means (6), said controller (24) being configured for data communication with said measuring means (5), Each of said cell culture chambers (1A-1C) includes a plurality of fluid inlets and outlets (10.1A-10.4A, 10.1B-10.4B, 10.1C-10.4C); all of said plurality of cell culture chambers (1A-1C) have different internal volumes; The controller (24) determines, based on the data measured by the measuring means (5), to operate said adjusting means (6) and / or said fluid flow control means (3: 3.1-3.9) are further configured to operate so that the medium is circulated through the cell culture chamber (1A-1C) containing the cells for the time required for the cells to be transferred to another cell culture chamber (1A-1C); The cells contained in the medium in the first chamber (1A) are transferred into a second chamber (1B) having an internal volume larger than the internal volume of the first chamber (1A) and then transferred sequentially into the chambers having larger internal volumes until the cell culture process is completed; The cell culture system further comprises a cell retention means configured to retain cells in the cell culture chamber (1A-1C).

2. 2. The system according to claim 1, characterized in that the cell retention means are filtration means (2) arranged inside the cell culture chambers (1A-1C) and configured to filter the culture medium and further configured to retain cells inside each cell culture chamber (1A-1C).

3. The filtering means (2) of at least one of the cell culture chambers (1A-1C) filter the cell culture chamber (1A-1C) by: a first compartment (14: 14A-14C) suitable for containing a culture medium therein; a second compartment (15: 15A-15C) adapted to receive therein cells contained in a culture medium; and 3. The system according to claim 2, characterized in that the filter membrane (12) is configured to allow the passage of the culture medium between both compartments (14: 14A-14C, 15: 15A-15C) and to retain the cells in the second compartment (15: 15A-15C).

4. 4. The system according to claim 2, wherein the filtering means (2) of at least one of the cell culture chambers (1A-1C) comprises at least one filter arranged at at least one of the fluid inlets (10.1-10.4) of the cell culture chamber.

5. said holding means comprising a pump (4) configured to circulate culture medium through said chambers (1A-1C) at a predetermined speed and flow direction such that the culture medium circulated through said chambers (1A-1C) is located at a first height (h1) relative to a bottom surface of said chambers (1A-1C); the cells contained in the medium in the cell culture chamber (1A-1C) are located at a second height (h2) relative to a bottom surface of the chamber (1A-1C); The first height (h1) is greater than the second height (h2); The system of claim 1, characterized in that when the culture medium circulates through the chambers (1A to 1C), the cells are prevented from being entrained out of the chambers (1A to 1C) more quickly than necessary.

6. 6. The system according to claim 1, wherein the conditioning means (6) comprises a conditioning chamber (13) configured to accommodate a gas therein, the conditioning chamber (13) being connected to the cell culture chambers (1A-1C) via a gas-permeable membrane (22).

7. The medium adjustment means (6) a conditioning chamber (13) connected to the plurality of channels and configured to accommodate a culture medium therein; A system according to any one of the preceding claims, characterized in that it comprises means (20) for injecting at least one gas into said conditioning chamber (13).

8. a medium reservoir (7) connected to said plurality of channels and configured to be in fluid communication with the interior of said cell culture chambers (1A-1C); and / or an initial cell reservoir (8) connected to said plurality of channels and configured to be in fluid communication with the interior of said cell culture chambers (1A-1C); and / or a retentate reservoir (9) connected to said plurality of channels and configured to receive therein retentate fluid originating from said cell culture chambers (1A-1C); and / or The system according to any one of claims 1 to 7, characterized in that it comprises a final product reservoir (11) connected to the plurality of channels and configured to be in fluid communication with the interior of the cell culture chambers (1A-1C).

9. The system according to any one of claims 1 to 8, characterized in that it comprises a pre-chamber, preferably a cell transfection pre-chamber, connected to the first cell culture chamber (1A).

10. The measuring means (5) connected to said plurality of channels; and / or The system according to any one of claims 1 to 9, characterized in that it is integrated inside the cell culture chamber (1).

11. The measuring means (5) a biomass sensor, and / or The system according to any one of claims 1 to 10, characterized in that it comprises an oxygen sensor, a pH sensor and / or a CO2 sensor.

12. The system according to any one of claims 1 to 11, characterized in that it is a suspension cell culture system suitable for containing cells suspended in a culture medium in said chambers (1A to 1C).

13. A system according to any one of claims 1 to 11, characterized in that it is a cell culture system with attachment of cells to microparticles or surfaces.

14. 1. A method for culturing cells, comprising: Implemented by a suspension cell culture system according to any one of claims 1 to 13, a) introducing cells contained in a culture medium into a first cell culture chamber (1A); b) circulating said medium through said channels, said medium being circulated through said first cell culture chamber (1A) for a period of time necessary for cells to be transferred to a second cell culture chamber (1B); c) transferring the cells contained in the medium into the second cell culture chamber (1B); d) repeating step b) inside the second cell culture chamber (1B); e) obtaining the cultured cells; said method further comprising monitoring the state of the cell culture and / or cells by means of said measuring means (5) in order to determine the point in time when said cells have acquired a particular property and when, according to step c), they must be transferred to a cell culture chamber (1B) having a larger internal volume compared to the cell culture chamber (1A) in which they are located and successively transferred to chambers having a larger internal volume.

15. 15. The method according to claim 14, characterized in that it comprises adjusting the culture medium by means of the adjusting means (6) if the measuring means (5) determines that the culture medium is in a deficiency state, preferably in a deficiency state of gas and / or nutrients.

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