WO2025141177A1

WO2025141177A1 - Recycling media in suspended fluid bed reactor

Info

Publication number: WO2025141177A1
Application number: PCT/EP2024/088597
Authority: WO
Inventors: Nicolaas Willem DE JONGH
Original assignee: Supreme
Current assignee: Supreme
Priority date: 2023-12-28
Filing date: 2024-12-27
Publication date: 2025-07-03
Anticipated expiration: 2026-06-28

Abstract

The invention relates to a cell culture system (1) comprising: A bioreactor (100), comprising a vessel (110) and a recycling outlet (120), said recycling outlet (120) being positioned in the vessel (110) so as to be able to extract, from the vessel (110), a multiphase flow comprising a liquid phase and a gas phase; a separator (300) receiving the multiphase flow, said separator (300) being configured to accommodate and maintain a multiphasic system, comprising both the liquid phase and the gas phase; and a recycled media transfer line (220) adapted for routing of at least a portion of the liquid phase from the separator (300) to the bioreactor (100). The invention also relates to a cell culture method (1000) using a cell culture system (1) of the invention and cell biomass obtained thereof.

Description

RECYCLING MEDIA IN SUSPENDED FLUID BED REACTOR

Field of the invention

[1] The present invention relates to the field of cell culture systems and their optimization. In particular, the invention relates to the field of advanced bioreactor technology, aimed at enhancing the efficiency of cell culture processes. This invention can provide a cell culture system for industrial scale production, offering high-density cultivation and efficient nutrient distribution for optimal growth and productivity.

Description of Related Art

[2] The cultivation of cells in bioreactors for the production of biological materials, such as proteins, vaccines, cultivated meat and cultivated leather; for drug discovery; and for tissue engineering; is a cornerstone of modern biotechnology. This process often involves the use of high-value materials, including algorithmic optimized cell culture media (Zhou, Tianxun et al. “A review of algorithmic approaches for cell culture media optimization.” Frontiers in bioengineering and biotechnology vol. 11 1195294. 11 May. 2023). The efficient use of these materials is crucial for economic viability, yet current methods often lead to significant waste. Indeed, traditional cell culture processes, including both fed-batch and perfusion systems, face challenges in managing the accumulation of waste byproducts produced by cells in culture media, such as ammonia, lactate, toxins or salts, which can hamper cell growth, promote unwanted differentiation, negatively impact cell viability, leading thus to poorer yield.

[3] The production of cultivated meat is particularly affected by this situation. Cultivated meat as a sustainable alternative to traditional intensive meat production confronts significant challenges, predominantly in cell culture systems and culture media management and optimization. Cultivated meat bioreactors should ensure consistent cell growth and quality, while also navigating the safety and regulatory requirements necessary for market viability (Jara, T.C., Park, K., Vahmani, P. et al. “Stem cell-based strategies and challenges for production of cultivated meat.” Nature Food 4, 841-853. October. 2023). Regarding the culture media, differences in glucose utilisation and amino acid consumption rates among various cell types like primary embryonic chicken muscle cells and murine C2C12 myoblasts highlight the need for adaptation of bioreactors and from the management of culture environments to the production of cultured meat (O’Neill, E.N., Ansel, J.C., Kwong, G.A. et al. Spent media analysis suggests cultivated meat media will require species and cell type optimization, npj Sci Food 6, 46. 2022). [4] In continuous flow bioreactor systems, a continuous flow of fresh media is provided while waste-loaded media is removed. This approach, while effective in maintaining optimal growth conditions, results in the elimination of media that still contains valuable nutrients. The high cost of culture media life cycle significantly contributes to the overall cost of cell production systems. Thus, an important challenge in cell culture technologies, including fluidized bed systems, is the efficient use of cell culture media.

[5] Some have proposed using cell culture waste for the growth of microorganisms (Lynch, Clara D., O’Connel, David J. “Conversion of mammalian cell culture media waste to microbial fermentation feed efficiently supports production of recombinant protein by Escherichia coli” PLOS ONE, 4 May. 2022). However, this maintains high production costs for cell growth and does not optimize the use of complex culture media dedicated to cell cultures. For instance, international patent publication WO 2023/156933 describes a system for rejuvenating a cell culture medium using an electric field to remove waste molecules. Another international patent publication WO 2022/238867 discloses means for acidifying and nanofiltrating a waste-loaded medium in order to rejuvenate it.

[6] Despite advances in techniques such as medium rejuvenation and selective waste removal, these methods often require complex and costly setups, and they are not always efficient in retaining critical nutrients in the culture medium. The conventional approach of continuous replacement of culture media in these systems leads to a substantial wastage of high-value materials. The current state of cell culture technology, therefore, presents a paradox. On one hand, the demand for high-efficiency biological production systems is increasing, driven by needs in healthcare, food technology, and other sectors. On the other hand, the existing cell culture methodologies are hampered by inefficiencies related to media use and waste management. These inefficiencies not only elevate production costs but also impact the scalability of these systems, particularly in applications requiring large volumes of culture media, such as the production of cultured meat or large-scale vaccine manufacturing or any animal cell-based products manufacturing.

[7] Thus, there is a significant need for an improved approach to cell culture that minimises the waste of valuable materials like cell culture media for large-scale cell production. Such an approach would ideally enhance the efficiency of nutrient utilisation, reduce the environmental footprint of bioprocessing, and lower the overall cost of production, thereby making cell-based products more accessible and sustainable (e.g. drugs, cultivated meat or cultivated leather).

Summary of the Invention

[8] The following sets forth a simplified summary of selected aspects, embodiments and examples of the present invention for the purpose of providing a basic understanding of the invention. However, this summary does not constitute an extensive overview of all the aspects, embodiments and examples of the invention. Its sole purpose is to present selected aspects, embodiments and examples of the invention in a concise form as an introduction to the more detailed description of the aspects, embodiments and examples of the invention that follow the summary.

[9] The invention aims to overcome the disadvantages of the prior art. In particular, the invention proposes a cell culture system comprising: a bioreactor, comprising a vessel and a recycling outlet, said recycling outlet being positioned in the vessel so as to be able to extract, from the vessel, a multiphase flow comprising a liquid phase and a gas phase; a separator receiving the multiphase flow, said separator being configured to accommodate and maintain a multiphasic system, comprising both the liquid phase and the gas phase; and a recycled media transfer line adapted for routing of at least a portion of the liquid phase from the separator to the bioreactor, in particular to the vessel.

[10] Such a cell culture system allows recycling of the media used in cell production. The use of a system capable of collecting multiphase flow, maintaining a multiphasic system and routing of at least a portion of the liquid phase from the separator to the bioreactor, is particularly adapted when dealing with a high-volume bioprocessing unit. Moreover, as it is described hereafter, the system according to the invention comprises features designed to enable scalability from lab-scale to large-scale.

[11] Hence, the present invention can be used to reduce production costs particularly in applications requiring large volumes of culture media, such as the production of cultured meat at industrial level or large-scale vaccine manufacturing.

[12] According to other optional features of the cell culture system according to the invention, it can optionally include one or more of the following characteristics alone or in combination: it further comprises an oxygen diffuser arranged to create interfaces, preferably thin film interfaces, between the liquid phase and the gas phase. This enhances the efficiency of nutrient utilisation and reduce the environmental footprint of bioprocessing. the oxygen diffuser is selected among: a sprayer, a T connector, a loose tube with an open endpoint, or an adaptor to dispense liquid against a surface without gas entrainment or foaming; the oxygen diffuser is a spraying system capable of spraying the multiphase flow in the separator; the bioreactor is selected from: a flow-controlled bioreactor, a stirred-tank bioreactor, wave/orbital shaking bioreactor, airlift bioreactor, fluidised bed bioreactor, packed bed bioreactor, and hollow-fibre bioreactor; the bioreactor is a fluidized bed bioreactor; it is configured to handle more than 1000 liters of culture volume with one or more bioreactors. This open the way for large scale production. the recycling outlet comprises an opening positioned within the vessel at a distance from the internal upper face of the vessel that is of at most 20 % of the total height of the vessel. This enhances the efficiency of nutrient utilisation and reduce the environmental footprint of bioprocessing. the separator is selected among: flash drum, flash drum with condenser, cyclone with condenser, sedimentation tank, sedimentation tank with condenser, or membranebased liquid-gas separator; the recycled media transfer line is arranged to connect a liquid section of the separator, preferably a solids-free liquid section of the separator, to a fresh media inlet of the vessel, preferably directly or indirectly. For example, the recycled media transfer line is arranged to connect a liquid section of the separator to a distribution plate underneath the vessel. Also, it can further comprise a mixing device configured to mix the fresh media with the recycled media. This enhances the efficiency of nutrient utilisation and reduce the environmental footprint of bioprocessing. it is configured to adjust the flow rate from a fresh transfer line and the recycled media transfer line in order to induce in the vessel a flow rate ensuring a fluidisation of the cell particles in the cell catalyst bed (e.g. maintaining an appropriate cell bed height and enabling continuous flow operation at an adequate cell growth rate). The combined flow rate, from the fresh transfer line and the recycled media transfer line can for example be of at least 1% in volume of the vessel per hour. This enhances the efficiency of nutrient utilisation and reduce the environmental footprint of bioprocessing. it is configured to adjust the flow rate from a fresh transfer line and the recycled media transfer line in order to induce in the vessel a flow rate of at least 1% of the vessel working volume. This enhances the efficiency of nutrient utilisation and reduce the environmental footprint of bioprocessing. it further comprises a recycled gas transfer line adapted for routing at least part of the gas phase from the separator in the bioreactor (directly or not). In particular it can be done by routing of the recycled gas via a T-connection with the fresh feed gas, after which this gas mixture enters the bioreactor, specifically through the center of the internal oxygenation devices; it further comprises a fresh media transfer line adapted for routing a fresh media in the vessel. it further comprises at least one gas exchange device protruding in the vessel. This enhances the efficiency of nutrient utilisation and reduce the environmental footprint of bioprocessing.

[13] According to another aspect, the present invention can also relate to a method for cell culture, using a cell culture system comprising a bioreactor, said bioreactor comprising: a vessel and a recycling outlet, a separator, and a recycle media transfer line; said method comprising:

- a step of extracting, from the vessel, a multiphase flow comprising a liquid phase and a gas phase;

- a step of transferring the multiphase flow to the separator and maintaining within the separator a multiphasic system, comprising both the liquid phase and the gas phase; and

- a step of routing of at least a portion of the liquid phase from the separator to the bioreactor, through the recycled media transfer line.

[14] According to another aspect, the present invention can also relate to a cell biomass obtainable from a method according to the invention, said cell biomass comprising less than 50 ppm of shear-stress protectant agent.

[15] According to yet another aspect, the present invention relates to an edible food product obtainable from cell biomass according to the invention, said edible food product according to the invention comprises less than 45 ppm of shear-stress protectant agent.

Brief description of the drawings

[16] The foregoing and other objects, features and advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which:

Figure 1 is a schematic view of a cell culture system according to an embodiment of the invention.

Figure 2 is a schematic view of a method of cell culture according to an embodiment of the invention.

[17] Several aspects of the present invention are disclosed with reference to flow diagrams and/or block diagrams of methods, devices and systems.

[18] On the figures, when present, the flow diagrams and/or block diagrams show the architecture, the functionality and possible implementation of devices or systems or methods, according to several embodiments of the invention.

[19] In some implementations, the functions associated with the box may appear in a different order than indicated in the drawings. For example, two boxes successively shown, may be performed substantially simultaneously, or boxes may sometimes be performed in the reverse order, depending on the functionality involved.

Detailed description

[20] A description of example embodiments of the invention follows.

[21] As used herein, the expressions “cultivated cells” or “cultured cells” are used interchangeably. They can refer to cells multiplied, differentiated, undifferentiated and/or grown, preferably in a controlled environment, using a culture medium. It refers in particular to cells with a growth controlled by mankind, for example in an industrial process, as opposed to cells from conventional meat are multiplied in a living organism or cells grown in a natural environment (e.g. forest grown mushrooms). Cultivated cells can refer to any cells or cell types belonging to Animalia kingdom for the proteins but also to Bacteria, Viridiplantae and Fungi kingdoms for example to provide additional proteins or fat. For example, the cultivated cells can be avian, fish or mammalian. Cultivated cells can originate from cells of any origin such as cells from biopsies, from stem cells isolated from animal embryos, or correspond to stem cells themselves. Cells can be cultivated as single cells, cell clusters, organoids, spheroids, or on microcarriers.

[22] As used herein, the expression “waste” or “waste products” are used interchangeably. They can refer to materials, byproducts, and/or molecules produced by the cultured cells that inhibit or prevent cell viability, growth and/or productivity, or that induce apoptosis, or are not useful to the cells, but not necessarily malignant. Moreover waste could impair differentiation or even trigger unwanted differentiation of the cultured cells. The waste can refer to, but is not limited to, e.g. ammonia, lactate, cell debris, toxins or salts, that accumulates in the culture media and/or gas.

[23] The terms “media” or “medium” are used interchangeably. They can refer, within the meaning of the invention, to a liquid, a growth medium, a culture medium, or an environment allowing the growth, proliferation, differentiation and maintenance of microorganisms and/or cells. Hence, the media can include nutrients. In other embodiments, the media can also include substance, small molecule or compound inducing or controlling the differentiation of the cultivated cells. [24] The term “nutrient” can refer, within the meaning of the invention, to any substance, small molecule or compound that provides nourishment essential for the maintenance of life and/or for growth. The term nutrient broadly comprises both macronutrients and micronutrients. These encompass essential nutrients like amino acids, vitamins, minerals, proteins, carbohydrates, fats and/or oxygen.

[25] The expressions “recycled medium” or “recycled media” can refer, within the meaning of the invention, to a culture medium or a culture media previously used and reinjected within the system according to the invention.

[26] The term “vessel” can refer, within the meaning of the invention, to a container or chamber designed to host and maintain biological reactions, particularly for the cultivation of animal cells. This vessel is typically part of a larger bioreactor system and is engineered to provide an optimal environment for cell growth, eventually differentiation and proliferation within a cultivation volume. The vessel working volume (or cultivation liquid volume) can refer to the vessel internal volume subtracted by the nutrient unit(s) volume and the eventual gas headspace. It generally corresponds to the volume of liquid inside the working vessel. The vessel cell bed volume can refer, when considering fluidized bed reactors, to the volume occupied by the cells during growth conditions. It should be about 90 % of the liquid volume.

[27] The expression “gas permeability” can refer, within the meaning of the invention, to the ability of a material to allow gases to pass through it. The gas permeability measures the ability of gases like oxygen and carbon dioxide to diffuse efficiently into and out of the culture medium. The gas permeability of a material is usually quantified by measuring the rate at which a specific gas passes through a material under a defined set of conditions, such as temperature and pressure.

[28] The expression “gas exchange” can refer, within the meaning of the invention, to the process by which gases are transferred for example across an element. This gas exchange element, comprising generally a gas exchange surface. Gas exchange, for example across membranes, typically occurs via diffusion. This means that gases move from an area of higher concentration to an area of lower concentration. The rate of diffusion usually depends on the concentration gradient across the element (e.g. membrane), the permeability of the membrane to each gas, and the physical properties of the gases themselves.

[29] The expression “biological products” can refer, within the meaning of the invention, to any products derived from living organisms or cells, encompassing cells themselves, cell biomass, and cell products. Hence, it can include a wide range of products such as vaccines, allergenics, cells, gene therapy, tissues, and recombinant therapeutic proteins. Biologies can be composed of sugars, proteins, or nucleic acids or complex combinations of these substances, or may be living entities such as cells or tissues.

[30] The term “a” or “an” as used herein can refer to "one or more" unless explicitly stated otherwise.

[31] As mentioned, the large-scale cell production such as animal cells for cultivated meat, cultivated leather or large-scale vaccine manufacturing requires large volumes of culture media and could largely benefit from culture media recycling.

[32] The inventors have developed a new cell culture system comprising parts specifically designed to reduce the production costs by allowing recycling of the culture media.

[33] Oxygen stands as a crucial nutrient in cell culture, yet its distribution poses challenges in large-scale cultivation. Moreover, the mass transfer characteristics at this scale often result in uneven or insufficient oxygen concentrations within the growth medium. While enhancing agitation in the growth area could facilitate better oxygen diffusion, this approach leads to unwanted shear stress causing cellular damage. Consequently, balancing oxygen supply without compromising cell integrity is a key consideration in optimising large-scale cell culture environments.

[34] The inventors have created a media recycling system designed to efficiently oxygenate the recycled media at a reduced cost. This innovation, especially suited for fluidized bed bioreactors, paves the way for large-scale cell production that is economically feasible.

[35] Hence, according to a first aspect, the invention relates to cell culture system 1. Advantageously, a cell culture system 1 according to the invention is adapted for growing cells, in particular animal cells. Preferably, a cell culture system 1 according to the invention is adapted for growing mammalian, avian or fish cells.

[36] A system 1 according to the invention comprises a bioreactor 100, a separator 300 being configured to accommodate and maintain a multiphasic system, comprising a liquid phase and a gas phase; and a recycled media transfer line 220 adapted for routing of at least a portion of the liquid phase from the separator 300 to the bioreactor 100.

[37] In particular, the system 1 according to the invention can comprise other distribution transfer lines 200; oxygen diffuser 350; fresh media tank 400; monitoring and adjustment devices 500; gas exchange devices 600 protruding in the vessel 110; separation devices 700; harvesting devices 800; and waste media tank 900. [38] As illustrated in Figure 1 , a system 1 according to the invention comprises a bioreactor 100. The system 1 according to the invention can comprise only one bioreactor 100 or several bioreactors 100.

[39] A bioreactor 100 in a system 1 according to the invention is preferably configured to allow cell culture, preferably animal cells, more preferably avian, mammalian or fish cells. In particular, it can be arranged to allow culture of cells in suspension, for example as a fluidized bed.

[40] The cell culture system 1 according to the invention can comprise a bioreactor 100 selected from: a flow-controlled bioreactor, an airlift bioreactor, a stirred-tank bioreactor, a wave/orbital shaking bioreactor, a fluidised bed bioreactor, a packed bed bioreactor, or a hollow-fibre bioreactor. When the system 1 according to the invention comprises at least two bioreactors 100, the bioreactors 100 can be a combination thereof. Preferably, the bioreactor 100 is a fluidized bed bioreactor.

[41] A bioreactor 100 comprises a vessel 110 and ports for supply and collection of media such as a recycling outlet 120.

[42] A bioreactor 100 can also comprise sampling ports 160 and harvesting ports 180.

[43] One of the advantages of the present invention is to allow large-scale culture of cells with reasonable cost.

[44] A bioreactor 100 and in particular a vessel 110 can include mechanical agitation means for example configured to fluidize the cell bed. However, contrary to the knowledge of those skilled in the art, the present invention, preferably, does not include mechanical agitation means in the vessel 110 of the bioreactor 100 for example configured to fluidize the cell bed. Hence, preferably, the working volume of the vessel 110 does not comprise impellers, or stirrers.

[45] As illustrated in Figure 1, the bioreactor 100 according to the invention comprises a vessel 110.

[46] As mentioned, one of the advantages of the present invention is to allow large-scale cell production with reduced waste of resources, including nutrients.

[47] Large-scale cell production using the technical solutions of the present invention can be reached through at least two routes.

[48] In an embodiment, the vessel 110 used for the growth of the cells is very large. Hence, the vessel 110 can have a working volume of at least 100 liters, preferably at least 5 000 liters, more preferably at least 30 000 liters, even more preferably at least 100 000 liters. For example, the invention can be suited to vessels having a working volume of at least 1 000 000 liters.

[49] In another embodiment, the vessel 110 used for the growth of the cells is not large but the system for cell cultivation comprises a large number of vessels 110 operating for example in parallel. The vessels 110 may be supplied, controlled and/or monitored independently in order to avoid contamination leading to loss of cultivated biomass. Hence, in this embodiment, the vessel 110 can have a working volume of at least 50 liters, preferably at least 100 liters, more preferably at least 500 liters, even more preferably at least 1 000 liters, even more preferably at least 2 000 liters. For example, the invention can be suited to vessels 110 having a working volume of at least 5 000 liters.

[50] As the system 1 according to the invention is adapted for large scale production, it is usually adapted to manage a cultivation liquid volume of at least 1 000 liters, more preferably at least 5 000 liters, more preferably at least 15 000 liters, even more preferably at least

50 000 liters. Given the two routes described here before, a system 1 according to the invention can typically comprise more than 100 vessels 110 and/or can comprise vessel 110 with working volume of more than 100 liters.

[51] Depending on the configuration, the vessel 110 used in the invention can have variable height. For example, it can have a height of at least 50 centimeters, preferably at least 1 meter, more preferably at least 2 meters, even more preferably at least 5 meters. For example, the invention can be adapted to vessels having a height of at least 10 meters. For example, the invention can be adapted to vessels having a height of 60 meters.

[52] The vessel 110 can have a diameter of at least 10 centimetres, preferably at least 50 centimeters, more preferably at least 1 meter, even more preferably at least 10 meters.

[53] Also, a bioreactor 100 in a system 1 according to the invention comprises a recycling outlet 120.

[54] The recycling outlet 120 is preferably positioned in the vessel 110 so as to be able to extract, from the vessel 110, a multiphase flow comprising a liquid phase and a gas phase.

[55] The multiphase flow extracted from the vessel 110 through the recycling outlet 120 is a flow constituted by sometime liquid, sometime gas, sometime both. The nature of the multiphase flow can change in the minute range, preferably in the second range and more preferably in the millisecond range. For example, in a time frame of less than one minute the recycling outlet 120 allows the exit of gas and liquid from the vessel 110. More preferably, the system 1 according to the invention is arranged in such a way that it allows the exit of gas and liquid from the vessel 110, through the recycling outlet 120, in a time frame of less than 10 seconds, more preferably in a time frame of less than 100 milliseconds.

[56] The recycling outlet 120 can have an opening in the vessel 110 whose section surface is of at least 0.75 mm², preferably at least 75 mm², more preferably at least 450 mm², even more preferably at least 7 500 mm².

[57] When the recycling outlet 120 has an opening in the vessel 110 whose section is a disk, it can have an opening in the vessel 110 of at least 1 mm diameter, preferably at least 10 mm, more preferably at least 25 mm, even more preferably at least 100 mm.

[58] The recycling outlet 120 opening is preferably positioned in an upper part of the vessel 110. The upper part of the vessel 110 can be the upper half or the upper quarter of the vessel 110. The recycling outlet 120 is more preferably positioned at a short distance from the upper wall of the vessel 110. However, the distance as such will be highly dependent on the height of the vessel 110. Hence, the recycling outlet 120 opening can be positioned within the vessel 110 at a distance from the internal upper face of the vessel 110 that is of at most 20% of the total height of the vessel 110, preferably at most 15%, more preferably at most 10%, even more preferably at most 5%.

[59] For example, the recycling outlet 120 opening can be positioned within the vessel 110 at a distance of at most 100 mm from the internal upper face of the vessel 110, preferably at most 50 mm, more preferably at most 10 mm, even more preferably at most 1 mm.

[60] As illustrated in Figure 1 , the system 1 according to the invention can comprise distribution transfer lines 200. These distribution transfer lines 200 are designed to circulate the culture media or gas within the bioreactor 100, ensuring uniform distribution of cells, nutrients, gases, and heat. This is achieved through a controlled circulation process, which can be facilitated by internal or external loops. Relevant components of these recirculation systems include pumps for fluid movement, impellers or agitators for mixing, and baffles to prevent vortex formation and ensure effective mixing. The system 1 is designed to operate under aseptic conditions to avoid contamination, an important aspect in cell culture bioreactors.

[61] Some of the distribution transfer lines 200 are described hereafter in association with the component they are interacting with.

[62] As illustrated in Figure 1 , the recycling outlet 120 can be connected to a recyclable media transfer line 210 arranged to transport the multiphase flow from the vessel 110 to the different parts of the systems involved in recycling.

[63] In particular, the recyclable media transfer line 210 can be arranged to transport the multiphase flow directly or indirectly to a separator 300.

[64] Indeed, as illustrated in Figure 1, a system 1 according to the invention comprises a separator 300. The separator 300 is preferably arranged to receive the multiphase flow.

[65] In particular, the separator 300 is configured to accommodate and maintain a multiphasic system, comprising both the liquid phase and the gas phase.

[66] The multiphasic system comprises the liquid phase, the gas phase and it can also comprise a solid phase. Indeed, in some configurations, cells cultivated in the vessels 110 can go through the recycling outlet 120.

[67] The separator 300 and the collection of the multiphase flow participate in the oxygenation of the collected media and thus to an increase of the oxygen concentration in the recycled media to be reinjected in the vessel 110.

[68] The separator 300 can be selected among: flash drum, flash drum with condenser, cyclone with condenser, sedimentation tank, sedimentation tank with condenser, or membrane-based liquid-gas separator.

[69] The separator 300 has an internal volume of at least 0.001 m³, preferably of at least 0.01 m³, more preferably of at least 0.1 m³, even more preferably of at least 0.5 m³.

[70] The separator 300 has an internal volume of at most 50 m³, preferably of at most 10 m³, more preferably of at most 5 m³, even more preferably of at most 1 m³.

[71] Advantageously, the separator 300 can comprise an anti-foaming element, preferably the anti-foaming element is selected among: liquid-gas separation membrane, a sponge-like mesh, packing material, nozzle redirecting liquid onto a flat surface.

[72] Advantageously, the system 1, comprises an oxygen diffuser 350 arranged to create interfaces between the liquid phase and the gas phase just before or inside the separator 300. These interfaces are preferably thin film interfaces.

[73] The oxygen diffuser 350 is preferably arranged to generate high surface exchange between the gas and the liquid phase. In particular, the oxygen diffuser 350 is arranged to generate an interfacial area of at least 100 m² /m³. Preferably, the oxygen diffuser 350 is arranged to generate an interfacial area of at least 250 m² /m³, more preferably at least 500 m²/m³, even more preferably at least 800 m²/m³. The interfacial area being measured in units of area per unit volume, such as square meter per cubic meter and being representative of the amount of gas-liquid interface available for mass transfer per unit volume of the oxygen diffuser 350.

[74] Advantageously, the oxygen diffuser 350 is arranged to generate a thin film of liquid phase or microdroplet of liquid phase. The thin film of liquid phase being for example a film with a thickness of less than 1 mm, preferably a film with a thickness of less than 0.5 mm, more preferably a film with a thickness of less than 250 pm. The microdroplets of liquid phase being for example liquid droplets of less than 100 pm in diameter, preferably less than 80 pm, more preferably less than 50 pm. The microdroplets diameter can be measured by laser diffraction or image analysis.

[75] The oxygen diffuser 350 can be selected among: a sprayer, a T connector, a loose tube with an open endpoint, or an adaptor to dispense liquid against the wall of a vessel 110 without gas entrainment or foaming. Preferably, the oxygen diffuser 350 is a spraying system capable of spraying the multiphase flow in a separator 300, said separator 300 being configured to accommodate and maintain a multiphasic system, comprising both a recyclable liquid phase and a gas phase.

[76] As illustrated in Figure 1 , a system 1 according to the invention can comprise a recycled media transfer line 220 adapted for routing of at least a portion of the liquid phase from the separator 300 to the bioreactor 100.

[77] In particular, the recycled media transfer line 220 can be adapted for routing of at least a portion of the liquid phase from the separator 300 to the vessel 110. Preferably, the recycled media transfer line 220 is connected from a solids-free liquid section of the separator 300 to a fresh media inlet of the bioreactor vessel 110. For example, the fresh media inlet can be positioned underneath a particle distribution plate of the bioreactor 100.

[78] The routing of the recycled liquid phase from the separator 300 to the bioreactor 100 or the vessel 110 can be direct. However, as an embodiment of the present invention, the routing of the recycled liquid phase from the separator 300 to the bioreactor 100 can be indirect.

[79] For example, when the recycled liquid phase is collected from the separator 300, it can be routed to a rejuvenating tank and/or a mixing tank before being reinjected in the vessel 110.

[80] The routing of media can be managed through distinct methods such as peristaltic pump, or the use of overhead gas pressure coupled with a control valve. The use of overhead gas pressure in combination with a control valve involves applying gas pressure to push the recycled media through the system 1. The flow rate and volume are regulated by adjusting the control valve, which modulates the pressure applied. This method can be particularly advantageous in systems in larger bioreactors where the use of pumps might be less efficient.

[81] A cell culture system 1 according to the invention can further comprise a spent media transfer line 290. This spent media transfer line 290 is preferably adapted for removing spent media from the separator 300.

[82] In particular, the spent media transfer line 290 can be arranged for removing spent media from the bottom of the separator 300. The spent media can be transported to a waste media tank 900 of the system 1. In another embodiment, the spent media can be transported to a harvesting device of the system 1 in case the spent media comprises cultivated cells.

[83] A cell culture system 1 according to the invention can further comprise a recycled gas transfer line 280. This recycled gas transfer line 280 is preferably adapted for routing recycled gas from the separator 300 to the vessel 110.

[84] The recycled gas transfer line 280 can be arranged to route the recycled gas to the vessel 110 directly or not. The introduction of the recycled gas is preferably done through a gas inlet. This can be used to maintain controlled environmental conditions within the vessel 110 while reducing the nutrient consumption of the cell culture.

[85] In addition, the design of the recycled gas transfer line 280 includes features that allow for precise control over the flow and composition of the recycled gas. As it will be described hereafter, the system according to the invention can be equipped with sensors and control mechanisms to monitor and adjust the gas composition, ensuring that the specific requirements of the cell culture are met. This level of control is preferred for processes where the gas composition significantly impacts cell growth and productivity.

[86] A cell culture system 1 according to the invention can further comprise a fresh media transfer line 240. This fresh media transfer line 240 is preferably adapted for routing fresh media from fresh media tank 400 to the vessel 110.

[87] The fresh media transfer line 240 can be arranged to route the fresh media to the vessel 110 directly or not. For example, the fresh media can be mixed with the recycled media before being introduced in the vessel 110. Hence, preferably, the system 1 further comprises a mixing device configured to mix the fresh media with the recycled media. Such a mixing device can be a stirred tank. Alternatively, the mixing can be done passively through a T, or through the injection of both media (fresh and recycled) in a lower part of the bioreactor where they will mix before entering the working volume of the vessel 110.

[88] In addition, the design of the fresh media transfer line 240 includes features that allow for precise control over the flow and composition of the fresh media. As it will be described hereafter, the system according to the invention can be equipped with sensors and control mechanisms to monitor and adjust the media composition, ensuring that the specific requirements of the cell culture are met. This level of control is preferred for processes where the media composition significantly impacts cell growth and productivity.

[89] Preferably, the system according to the invention is configured to adjust the flow rate from the fresh media transfer line 240 and the recycled media transfer line 220 in order to improve the mass transfer within the vessel 110.

[90] Indeed, a system according to the invention advantageously comprises a flow control system. Preferably, the flow control system is designed for enhancing mass transfer, ensuring efficient nutrient delivery and waste removal.

[91] Preferably, the flow control system is configured to continuously circulate medium through the vessel 110, enhancing mass transfer by allowing constant homogenization of the medium within the vessel 110.

[92] Typically, the control system is configured to continuously circulate medium through the vessel 110 at an entering flow rate corresponding to at least 1% of the vessel working volume per hour, preferably at least 2%, more preferably at least 3%, even more preferably at least 4 % of the vessel working volume per hour.

[93] While conventional bioreactor 100 will use sparging devices, agitation or stirring mechanisms, or microbubble systems, to enhance medium homogenization in the vessel 110, the present invention can be based in a high velocity culture medium circulation within the vessel 100, between one or at least two media inlets and one or at least two recycling outlets 120, to improve culture medium circulation and homogeneity.

[94] As large-scale applications are accessible with the current invention, the medium from within the vessel 110 can be removed from the vessel at a rate corresponding to at least 3 % of the volume of the vessel/h, more preferably at least 4 % of the volume of the vessel/h, even more preferably at least 5 % of the volume of the vessel/h.

[95] Subsequently, the extracted medium can undergo a rejuvenation process. This can be accomplished through an integrated rejuvenation system equipped with filtration, electrodialysis, and other separation technologies. Thus, the system 1 can be specifically designed to selectively remove waste products while simultaneously replenishing essential nutrients. This dual-action process ensures that the rejuvenated medium is restored to a condition that is conducive for cell growth.

[96] The recycled medium can be circulated back into the vessel 110. As mentioned, this is typically done using a system of pumps and controlled flow paths to ensure even distribution without disturbing the cells.

[97] Advantageously, the recycled medium can be subjected to an automated quality assessment. As described hereafter, this step involves evaluating the rejuvenated medium for optimal nutrient levels, appropriate pH balance, and the absence of harmful byproducts. The assessment uses state-of-the-art sensors and analytical tools to ensure that the culture medium meets stringent quality standards required for effective cell culture growth.

[98] The medium can be then recirculated back into the vessel 110. This recirculation can be carried out using a system of pumps and controlled flow paths. The design of this system is focused on ensuring an even distribution of the rejuvenated medium across the cell culture environment, while minimizing any potential disturbances to the cells. The flow paths are configured to maintain a gentle yet effective circulation pattern, catering to the delicate nature of the cell cultures.

[99] As illustrated in Figure 1 , a cell culture system 1 according to the invention can comprise a monitoring and adjustment system 500. Preferably, the monitoring and adjustment system 500 is configured for monitoring and adapting growth parameters such as pH, temperature, and nutrient concentration such as oxygen levels. Moreover the monitoring and adjustment system 500 can be configured for monitoring pressure within the bioreactor 100 and/or the gas exchange device 600, waste or waste metabolites level and other gasses such as CO2.

[100] Hence, the monitoring and adjustment system 500 can comprise sensors and control devices distributed throughout the cell culture system 1 to continuously monitor environmental parameters like pH, temperature, nutrient concentration including oxygen levels, pressure and waste or waste metabolites level. The collected data can be used to adjust conditions in real-time, ensuring uniformity in nutrient distribution for example.

[101] A system 1 according to the invention can comprise automated feedback loops that respond to sensor inputs, adjusting environmental conditions dynamically. This can include systems for automated pH adjustment, temperature control, and nutrient dosing. Preferably, the system 1 thus comprises devices configured to regularly monitor cell population using inline sampling and analytical techniques.

[102] In particular, the monitoring and adjustment system 500 can be configured to carry out a temperature control. Preferably, it is configured to maintain the vessel 110 at an ideal temperature for cell growth and/or to ensure cell viability. This could involve heating or cooling devices with sensors to detect and adjust the temperature as needed. This can be used for maintaining the bioreactor 100 at an ideal temperature conducive to cell growth and/or to ensure cell viability. The sensors are placed to continuously monitor the internal temperature of the bioreactor 100 or other components of the system 1 such as the separator 300. The monitoring and adjustment system 500 can be programmed to activate heating or cooling mechanisms as necessary to maintain the temperature within a predefined optimal range.

[103] In particular, the monitoring and adjustment system 500 can be configured to carry out a pH control. Preferably, it is configured to monitor the pH level of the culture medium, the recycled medium, the fresh medium and to adjust the pH for example using acidic or basic solutions. This automated adjustment ensures that the pH level remains within a range that is conducive to the viability and productivity of the cell culture.

[104] In particular, the monitoring and adjustment system 500 can be configured to carry out a nutrients level control. Preferably, it comprises sensors designed to measure the concentration of essential nutrients such as glucose, amino acids, and vitamins in the culture medium. Furthermore, it can be configured to trigger the addition of nutrients when levels fall below a set threshold. Preferably, the monitoring and adjustment system 500 is calibrated to recognize when nutrient levels fall below a predetermined threshold and triggers the addition of the required nutrients, thereby ensuring that the cells are consistently provided with the necessary components for growth and development.

[105] The monitoring and adjustment system 500 can be configured to control oxygen levels in the culture medium, ensuring cells receive adequate oxygen for metabolism. Preferably, it comprises dissolved oxygen sensors and/or gaseous oxygen designed to measure the concentration of oxygen in the liquid phase and/or the gas phase. Furthermore, it can be configured to trigger the addition of oxygen when levels fall below a set threshold. In particular, the monitoring and adjustment system 500 can be configured to control the pressure of the gaseous phase providing the oxygen supply. Indeed, the transport of oxygen at the gas exchange surface can scale with the external gas pressure.

[106] In particular, the monitoring and adjustment system 500 can be configured to carry out a waste level control. Preferably, it comprises sensors designed to measure the concentration of cell debris and waste such as ammonia, lactate, cell debris, toxins or salts, that accumulates in the culture media and/or gas. Furthermore, it can be configured to trigger the recycling of spent culture media or renewal with fresh culture media when levels increase above a set threshold. Preferably, the system is calibrated to recognize when waste levels increase above a predetermined threshold and trigger the recycling of spent culture media or renewal with fresh culture media, thereby ensuring that the cells are consistently thriving in an environment prone to growth and development.

[107] In particular, the monitoring and adjustment system 500 can be configured to carry out a pressure control system. Preferably, it is configured to maintain the vessel 110 at an ideal pressure for cell growth and/or to ensure cell viability. The sensors are placed to continuously monitor the internal pressure of the bioreactor 100 or other components of the system 1 such as the separator 300.

[108] As mentioned, all these sensors can be integrated in automated feedback loops configured to automatically adjust conditions within the vessel 110. Also, the cell culture system 1 according to the invention can be configured to log all sensor data for real-time monitoring and historical analysis, aiding in process optimization and quality control.

[109] As illustrated in Figure 1 , a system 1 according to the invention advantageously comprises at least one gas exchange device 600 protruding in the vessel 110. For example, a bioreactor 100 comprises at least two, at least five gas exchange devices 600, preferably at least ten gas exchange devices 600, more preferably at least twenty gas exchange devices 600, and even more preferably at least fifty gas exchange devices 600. In particular these gas exchange devices 600 are arranged so that at least part, preferably most of their gaseous exchange surface protrudes in the vessel 110 working volume.

[110] The gas exchange devices 600 can be useful for enhancing mass transfer, ensuring efficient nutrient delivery and waste removal. The gas exchange devices 600 preferably have a gaseous exchange surface in the vessel 110.

[111] Preferably, the gas exchange device 600 has a hollow cylindrical structure. More preferably, the hollow cylindrical structure has an aspect ratio of at least five, even more preferably at least ten. Even more preferably, the gas exchange device 600 comprises an hydrophobic gas-permeable wall, the gas-permeable wall having a gas permeability of at least 10 cm³/m².d.bar, preferably at least 50 cm³/m².d.bar, more preferably at least 100 cm³/m².d.bar and even more preferably at least 300 cm³/m².d.bar.

[112] The gas exchange devices 600 protruding in the vessel 110 are configured to diffuse oxygen within the vessel 110. It preferably involves the use of semi-permeable membranes that allow oxygen to pass through while keeping the culture media and cells contained outside the device 600.

[113] In particular, the gas exchange devices 600 protruding in the vessel 110 are configured to allow oxygen permeation through the membrane due to a concentration gradient between the gas phase (high oxygen) and the liquid phase (low oxygen).

[114] These oxygenation devices 600 induce a minimal shear stress compared to sparging, reducing the risk of cell damage, especially important for sensitive cell lines. Moreover, combined with the control of the medium flow within the vessel 110, such a method allows a decorrelation between oxygenation and shear stress.

[115] Typical gas flow rate can be of at least 0.1 vvm (volume of injected gas per media volume per minute).

[116] As illustrated in Figure 1 , a system 1 according to the invention can comprise separation devices 700. Preferably, the separation devices 700 are adapted to facilitate selective mass transfer, allowing efficient nutrient supply and waste removal at the cellular level.

[117] A system 1 according to the invention can comprise membrane filtration units 710. Semi-permeable membranes allowing the passage of culture medium while retaining cells can be used to prevent the passage of cells while allowing smaller molecules and waste products to pass through. Preferably, a membrane filtration unit 710 can be used on the recycled media transfer line 220 to ensure that the recycled medium injected in the vessel 110 will not comprise cells. These units 710 incorporate semi-permeable membranes to facilitate the selective passage of the culture medium while retaining cells. A distinguishing feature of these units is the pore size of the membrane, which can be chosen based on the average size of the cells being cultured. This sizing can allow the membrane to prevent the passage of cells, while permitting smaller molecules and waste products to filter through. The membranes are designed for optimal permeability and strength, ensuring minimal impact on the viability of the retained cells. [118] A system 1 according to the invention can comprise centrifugal separators 720. These devices 720 use centrifugal force to separate cells from the culture medium. The centrifugal force causes cells to aggregate and separate from the less dense medium. This method is particularly efficient for large-scale production. Preferably, centrifugal separators 720 can be used to isolate harvested cells from culture medium. The centrifugal separators 720 operate on the principle of centrifugal force to effectuate the separation of cells from the culture medium. When the culture medium is subjected to this force, cells, being denser, aggregate and separate from the less dense medium. This method is particularly advantageous for processing large volumes of culture medium, offering a scalable solution for cell retention. The separators 720 are calibrated to apply a specific centrifugal force, tailored to the density and size of the cells, ensuring efficient and gentle separation.

[119] As illustrated in Figure 1 , a system 1 according to the invention can comprise a harvesting system 800. Preferably, the harvesting system 800 is adapted for collecting biological products from large volumes without compromising quality and eventually asepsis.

[120] The harvesting system 800 can be a continuous or semi-continuous harvesting system 800 that allows for the regular collection of biological products preferably while maintaining the cell culture’s integrity.

[121] The harvesting system 800 can include separation technologies, like membrane filtration or centrifugation, integrated within the cell culture system 1.

[122] In an embodiment of the invention, under a continuous operating regime, where the harvesting line is positioned at a high cell concentration position, the outflow volumetric flow rate has a maximum value equal to the maximum specific growth rate characterised by the type of cell, multiplied by the total reactor volume.

[123] In another aspect, the invention relates to a method 1000 for cell culture, using a cell culture system according to the invention.

[124] In particular, a method 1000 for cell culture uses a cell culture system 1 comprising a bioreactor 100, said bioreactor comprising: a vessel 110 and a recycling outlet 120, a separator 300, and a recyclable media transfer line 210.

[125] As illustrated in Figure 2, the method 1000 according to the invention comprises: a step of extracting 1100, from the vessel 110, a multiphase flow comprising a liquid phase and a gas phase; a step of transferring 1200 the multiphase flow to the separator 300 and maintaining within the separator 300 a multiphasic system, comprising both the liquid phase and the gas phase; and a step of routing 1300 of at least a portion of the liquid phase from the separator 300 to the bioreactor 100.

[126] As described, the cell culture within the bioreactor 100 is closely monitored to determine the optimal time for biomass collection. The cell biomass is harvested when the cell culture reaches a desired density and viability status. [127] The harvesting can be done while the bioreactor 100 is operating or after that the fluidization has been paused. The cessation of the fluidizing elements allows for the cells to transition from a growth phase to a state that is more conducive to harvesting. For the collection of the biological products, the culture media comprising the cell biomass can be drained from the bottom of the vessel 110.

[128] Following the collection, biological products can be subjected to a centrifugation process. This step is designed to separate the cells from any remaining culture medium and to concentrate the cell biomass. The resulting cell pellet can then be subjected to a washing process using for example a sterile buffer solution. The purpose of this washing process is to remove any impurities or residual media components, further purifying the cell biomass.

[129] According to another aspect, the present invention can also relate to a biological product obtainable from a method 1000 according to the invention. Preferably, the present invention can also relate to a cell biomass obtainable from a method 1000 according to the invention. More preferably, the present invention can relate to a cell biomass obtained from a method 1000 according to the invention.

[130] Preferably, said biological product in particular cell biomass comprising less than 50 ppm of shear-stress protectant agent. More preferably, said biological product in particular cell biomass comprising less than 5 ppm of shear-stress protectant agent, even more preferably less than 0.5 ppm. Indeed, as the oxygen diffuser 1 according to the invention does not necessitate mechanical agitation of the culture media, a method 1000 according to the invention does not necessitate the use of shear-stress protectant such as anti-foaming agent.

[131] The shear-stress protectant agent can preferably be selected among biocompatible non-ionic surfactants or polymers configured to stabilise cell membranes to reduce the shear forces applied to cells, especially in environments with air or gas bubbles. Also, the shearstress protectant agent is recognized as a safe and effective ingredient in various food and pharmaceutical applications. In particular, the shear-stress protectant agent is selected among the antifoaming agents, such as silicone-based antifoamers, polypropylene glycols, or poloxamers.

[132] In particular, the shear-stress protectant agent is a polyoxyethylene-polyoxypropylene block copolymer such as a poloxamers. Preferably, the shear-stress protectant agent is a triblock copolymer composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Indeed, these compounds are known anti-foaming agents that can be used in cell cultivation. More preferably, the shear-stress protectant agent is a poloxamer (also called Pluronic®), such as Poloxamer 188. [133] According to yet another aspect, the present invention can also relate to an edible food product obtainable from cell biomass according to the invention. In particular the invention relates to an edible food product obtained from, and/or comprising, cell biomass according to the invention.

[134] Preferably an edible food product according to the invention comprises less than 45 ppm of shear-stress protectant agent. More preferably, said cell biomass comprising less than 2.5 ppm of shear-stress protectant agent, even more preferably less than 0.25 ppm.

[135] Advantageously, the edible food product according to the invention can be considered as an ingredient for an alternative to conventional meat products or as an alternative to conventional meat products as such.

[136] An edible food product according to the invention can for example be a ready-to-eat food product that can be consumed directly, or eventually after a processing step (e.g. freezing, crushing, squishing, braiding, cutting, grinding, mixing, shredding, squeezing, dosing, molding, pressing, 3D printing, extruding, baking or cooking steps such as smoking, roasting, frying, surface treatment, and/or coating) and/or a cooking step. An edible food product according to the invention can also be an intermediate product to be used in combination with other products to produce a ready-to-eat food component. In particular, the edible food product according to the invention can be an alternative product to the conventional meat which aims to mimic a conventional meat product (e.g. steak, sausage, pate, nugget, bacon...). An edible food product according to the invention can exhibit an improved meat-like texture and/or meat-like flavor compared to an edible meat alternative food product made from plant proteins.

[137] The edible food product according to the invention can be a finished product or an ingredient for food processing. Preferably, the edible food product according to the invention mimics a conventional animal-derived edible food product. The edible food product according to the invention can be a raw, pre-cooked or a cooked product. For example, the edible food product according to the invention is a cooked edible food product or a pre-cooked edible food product. For example, the edible food product is precooked to be further pan-fried. Alternatively, the edible food product is a raw product.

[138] The invention can be the subject of numerous variants and applications other than those described above. In particular, unless otherwise indicated, the different structural and functional characteristics of each of the implementations described above should not be considered as combined and I or closely and I or inextricably linked to each other, but on the contrary as simple juxtapositions. In addition, the structural and I or functional characteristics of the various embodiments described above may be the subject in whole or in part of any different juxtaposition or any different combination.

Claims

1. A cell culture system (1) comprising:

- A bioreactor (100), comprising a vessel (110) and a recycling outlet (120), said recycling outlet (120) being positioned in the vessel (110) so as to be able to extract, from the vessel (110), a multiphase flow comprising a liquid phase and a gas phase;

- A separator (300) receiving the multiphase flow, said separator (300) being configured to accommodate and maintain a multiphasic system, comprising both the liquid phase and the gas phase; and

- A recycled media transfer line (220) adapted for routing of at least a portion of the liquid phase from the separator (300) to the bioreactor (100).

2. The cell culture system (1) according to claim 1 , wherein it further comprises an oxygen diffuser (350) arranged to create interfaces, preferably thin film interfaces, between the liquid phase and the gas phase.

3. The cell culture system (1) according to claim 2, wherein the oxygen diffuser (350) is selected among: a sprayer, a T connector, a loose tube with an open endpoint, or an adaptor to dispense liquid against a surface without gas entrainment or foaming.

4. The cell culture system (1) according to claim 2 or 3, wherein the oxygen diffuser (350) is a spraying system capable of spraying the multiphase flow in the separator (300).

5. The cell culture system (1) according to anyone of claim 1 to 4, wherein the bioreactor (100) is selected from: a flow-controlled bioreactor, a stirred-tank bioreactor, wave/orbital shaking bioreactor, airlift bioreactor, fluidised bed bioreactor, packed bed bioreactor, and hollow-fibre bioreactor.

6. The cell culture system (1) according to anyone of claim 1 to 5, wherein the bioreactor (100) is a fluidized bed bioreactor.

7. The cell culture system (1) according to anyone of claim 1 to 6, wherein it is configured to handle more than 1000 liters of culture volume with one or more bioreactors (100).

8. The cell culture system (1) according to anyone of claim 1 to 7, wherein the recycling outlet (120) comprises an opening positioned within the vessel (110) at a distance from the internal upper face of the vessel (110) that is of at most 20 % of the total height of the vessel (110).

9. The cell culture system (1) according to anyone of claim 1 to 8, wherein the separator (300) is selected among: flash drum, flash drum with condenser, cyclone with condenser, sedimentation tank, sedimentation tank with condenser, or membranebased liquid-gas separator.

10. The cell culture system (1) according to anyone of claim 1 to 9, wherein the recycled media transfer line (220) is arranged to connect a liquid section of the separator (300), to a fresh media inlet of the vessel (110).

11. The cell culture system (1) according to anyone of claim 1 to 10, wherein it is configured to adjust the flow rate from a fresh transfer line (240) and the recycled media transfer line (220) in order to induce in the vessel (110) a flow rate ensuring a fluidisation of the cell particles in the cell catalyst bed.

12. The cell culture system (1) according to anyone of claim 1 to 10, wherein it is configured to adjust the flow rate from a fresh transfer line (240) and the recycled media transfer line (220) in order to induce in the vessel (110) a flow rate of at least 1% of the vessel working volume.

13. The cell culture system (1) according to anyone of claim 1 to 12, wherein it further comprises a recycled gas transfer line (280) adapted for routing at least part of the gas phase from the separator (300) in the bioreactor (100).

14. The cell culture system (1) according to anyone of claim 1 to 13, wherein it further comprises at least one gas exchange device (600) protruding in the vessel (110).

15. A method (1000) for cell culture, using a cell culture system (1) comprising a bioreactor (100), said bioreactor (100) comprising: a vessel (110) and a recycling outlet (120), a separator (300), and a recycled media transfer line (220); said method (1000) comprising: - A step of extracting (1100), from the vessel (110), a multiphase flow comprising a liquid phase and a gas phase;

- A step of transferring (1200) the multiphase flow to the separator (300) and maintaining within the separator (300) a multiphasic system, comprising both the liquid phase and the gas phase; and

- A step of routing (1300) of at least a portion of the liquid phase from the separator (300) to the bioreactor (100), through the recycled media transfer line (220).