System and method adapted for culturing animal cells at high-scale with low shear stress

Abstract

The invention relates to a bioreactor (100) adapted for culturing animal cells, comprising: a vessel (110) defining a cultivation volume (111); at least two gas exchange devices (120) protruding in the vessel (110), each gas exchange devices (120) having a gaseous exchange surface (121) in the vessel (110); and at least one media inlet (130) and at least one media outlet (135), said media inlet (130) and outlet (135) being configured to operate simultaneously. The invention also relates to methods, cell biomass obtained from these methods and edible food products including this biomass.

Description

SYSTEM AND METHOD ADAPTED FOR CULTURING ANIMAL CELLS AT HIGH- SCALE WITH LOW SHEAR STRESSField 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. The manufacturing of biological products predominantly employs stirred tank bioreactors or bioreactors operating with fluidized beds (US5601757) and operation involves three primary phases: expansion, production, and harvest, with each phase necessitating precise control of chemical concentrations and physical parameters. These traditional cell culture processes, including both fed-batch and continuous flow bioreactor systems, face challenges in managing the sufficient homogenization of the culture medium and the accumulation of waste byproducts, such as ammonia and lactate, which can inhibit cell growth and productivity (Yang, Ming et al. “Cell culture medium cycling in cultured meat: Key factors and potential strategies”, Trends in Food Science & Technology vol. 138 0924-2244. August. 2023).[3] The production of cultivated meat is particularly affected by this situation. Cultivated meat as a sustainable alternative to traditional meat production confronts significant challenges, predominantly in cell culture systems and culture media management. 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).[4] An important challenge in cell culture technologies, particularly in systems such as fluidized bed bioreactors, is the efficient oxygenation of the culture medium (Abdullah, Al-Ani et al. “Oxygenation in cell culture: Critical parameters for reproducibility are routinely not reported.” Pios One. 16 October. 2018). This challenge stems from the high metabolic demand of animal cells, such as mammalian, avian or fish cells, which require a constant and adequate supply of oxygen for optimal growth and productivity, particularly at high cell density. In fluidized bed systems, where cells are suspended in a fluid medium, achieving uniform oxygen distribution can be complex. The dynamic nature of these systems, characterized by the continuous movement of both cells and media, necessitates precise control over oxygen transfer rates. Insufficient oxygenation can lead to hypoxic conditions, adversely affecting cell growth and metabolism, ultimately compromising the yield and quality of the bioproduct or cell biomass itself. Overcoming the challenges of oxygenation in these systems is crucial for advancing cell culture technologies, particularly for large- scale and high-density cell cultivation.[5] Some have introduced the use of an oxygenator module added to a bioreactor, which enhances the oxygenation of bio-fabricated tissues during culture (Chen, A.M., Lashmet, M., Isidan, A. et al. “Oxygenation Profiles of Human Blood, Cell Culture Medium, and Water for Perfusion of 3D-Bioprinted Tissues using the FABRICA Bioreactor Platform.” Scientific Reports 10, 7237. April. 2020). The study explored the oxygenation of water, cell culture medium, and human blood under various conditions, demonstrating that the system could maintain stable oxygen levels essential for viable tissue cultures. In this laboratory scale study, the ability to move to high scale production has not been evaluated. However, it is possible to doubt the effectiveness of this solution when it will have to oxygenate thousands of liters of culture medium every minute.[6] Hence, despite these advances, the animal cell culture industry, e.g. for cultivated meat, cultivated leather or vaccine production, is still in demand for economical solutions for large-scale cell production. Indeed, 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 still hampered by inefficiencies related for example to insufficient oxygenation of the culture medium. 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 cultivated meat or large-scale vaccine manufacturing.[7] Thus, there is a significant need for an improved approach to cell culture that maximizes the oxygenation of the cell culture media for large-scale animal cells production in suspension. Such an approach enhances the efficiency of nutrient utilization,reduces the environmental footprint of bioprocessing, and lowers the overall cost of production, thereby making animal cell-based products more accessible and sustainable.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 bioreactor adapted for culturing animal cells, comprising: a vessel defining a cultivation liquid volume; at least two gas exchange devices protruding in the vessel, each gas exchange devices having a backbone and a gaseous exchange surface, said gaseous exchange surface being arranged to control gas exchange between a liquid phase flowing in the cultivation volume and a gaseous phase flowing in the gas exchange devices; and at least one media inlet and at least one media outlet, said media inlet and outlet being configured to induce a flow in the cultivation volume capable of preventing cells from sedimenting, preferably operating simultaneously.

[0010] A bioreactor according to the invention allows an improvement of the distribution of oxygen in a bioreactor. This improvement is crucial for cell growth, as it ensures cells receive the necessary oxygen uniformly. Moreover, this solution is designed to increase efficiency without increasing shear stress, which is necessary for maintaining cell integrity and viability at high scale. A key advantage of this invention is its ability to minimize shear stress during the culture process. Indeed, excessive shear stress can damage delicate cell structures, impeding growth and productivity.

[0011] The technology employed in this invention allows for gentle yet effective distribution of the oxygen, ensuring cell viability and integrity are maintained, which isessential for high-quality cell-based product development. Moreover, this improved distribution of the oxygen is combined with a configuration of the bioreactor allowing a management of the flow of culture medium in the bioreactor, controlled at the inlet and outlet ports of the culture medium allowing the cell bed to be kept in suspension.

[0012] These combined features produce a scalable oxygenation with height that can reduce or remove the need to use shear stress protectant compounds such as antifoaming agents. Hence, this invention provides an effective solution in particular for industrial-scale bioreactors. As a result, the present invention can be used to reduce production costs particularly in applications requiring large volumes of culture media, such as the production of cultivated meat or large-scale vaccine manufacturing.

[0013] According to other optional features of the bioreactor according to the invention, it can optionally include one or more of the following characteristics alone or in combination: the backbone is arranged so as not to deform under the action of the flow in the cultivation volume capable of preventing cells from sedimenting. This allows an improvement of the distribution of oxygen in a bioreactor. the cultivation liquid volume does not comprise component for mechanical agitation. This allows a reducing shear stress on cells. the gas exchange devices protruding in the vessel are configured to supply oxygen in an amount that is decorrelated from the shear-stress applied to the cells in the vessel. This allows an improvement of the distribution of oxygen in a bioreactor with reduced shear stress. a cumulated area of gaseous exchange surfaces within the vessel is arranged such that a value of a ratio of cumulated surface area of gaseous exchange surfaces within the vessel on the volume of the vessel is of at least 10 cm-1. This allows an improvement of the distribution of oxygen in a bioreactor. the gas exchange devices protruding in the vessel are configured to supply oxygen through a mechanism that do not modify significantly neither the velocity at the cell-liquid interface not the viscosity of the media. This allows an improvement of the distribution of oxygen in a bioreactor. it further comprises at least one gas inlet, preferably said at least one gas inlet being part of the gas exchange device; In particular, it further comprises at least one gas inlet and at least one gas outlet, said gas inlet and gas outlet beingarranged to permit a gas phase flow between them. This allows an improvement of the distribution of oxygen in a bioreactor. a ratio between the cumulated area of the media inlet openings to the gaseous exchange surface in the vessel is lower than 0.01. This allows an improvement of the distribution of oxygen in a bioreactor. the height of the vessel is at least 1 m. this open the way to large scale production. in the vessel, the gaseous exchange surface of each of the gas exchange devices is separated by at most 50 mm from the surface of the other gas exchange devices. This allows an improvement of the distribution of oxygen in a bioreactor. the gaseous exchange surface has a gas permeability of at least 10 cm3 / m2.d.bar.

[0014] According to another aspect, the invention can also relate to a method for culturing animal cells said method comprising the use of a bioreactor comprising a vessel defining a cultivation volume; at least two gas exchange devices protruding in the vessel, each gas exchange devices having a backbone structure and a gaseous exchange surface in the vessel; and at least one media inlet and at least one media outlet; said method comprising the following steps: adding culture media through the media inlet; removing culture media through the media outlet; exchanging gas at gaseous exchange surface between a gas fluid circulating within the gas exchange devices and the culture media in the cultivation volume; said media inlet and outlet operating to induce a flow in the cultivation volume capable of preventing cells from sedimenting, said gas exchange devices controlling the gas exchange at the gaseous exchange surface between a liquid phase flowing in the cultivation volume and a gaseous phase flowing in the gas exchange devices.

[0015] According to other optional features of the method according to the invention, it can optionally include one or more of the following characteristics alone or in combination: the flow rate of the culture media entering the vessel is selected to balance thegravitational force exerted on the cells which would cause them to sediment, preferably the linear velocity in the vessel is of at least 0.1 cm / hour; the flow rate of the culture media entering the vessel is of at least 1 % of the vessel working volume per hour. This allows an improvement of the distribution of oxygen in a bioreactor. the flow rate of the culture media entering the vessel is of at most 4% of the vessel liquid volume per hour. the superficial velocity of the culture media at the gaseous exchange surface is of at most 2 cm / h. the flow rate of the gas fluid circulating within the gas exchange devices is of at least 0.1 times the vessel liquid volume per minute; the pressure of the gas fluid circulating within the gas exchange devices is at least atmospheric. it comprises a monitoring step comprising: monitoring the fluid dynamics within the vessel; monitoring the height of the fluidized bed; monitoring the pH Levels; monitoring the temperature; monitoring the cell density; monitoring the nutrient concentration; and / or monitoring the pressure. In particular, it comprises a monitoring step comprising: monitoring the fluid dynamics within the vessel; monitoring the height of the fluidized bed; monitoring and adjusting the pH Levels; monitoring and adjusting the temperature; monitoring the cell density; monitoring and adjusting the nutrient concentration; and / or monitoring and adjusting the pressure. the flow rate of the culture media entering the bioreactor is calculated as a function of the values of the gas flow rate, the gas pressure within the gas exchange device, the gas permeability of the gas exchange membrane, the concentration of oxygen in the gas, the gas exchange surface area, and the vessel working volume; the flow rate of the culture media entering the bioreactor is further calculated based on the oxygen consumption of the cells in the vessel; the flow rate of the culture media entering the bioreactor is calculated according to the values of the gas flow rate, the gas pressure, the concentration of oxygen in the gas, the gas exchange surface area, and the vessel working volume.

[0016] According to another aspect, the present invention can also relate to a cellbiomass obtainable from a method according to the invention, said cell biomass comprising less than 50 ppm of shear-stress protectant agent (antifoaming agent).

[0017] According to yet another aspect, the present invention can also relate to an edible food product obtainable from cell biomass according to the invention, said edible food product comprising less than 45 ppm of shear-stress protectant agent.Brief description of the drawings

[0018] 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:FIG. 1 is a schematic view of a bioreactor adapted for culturing animal cells according to the present invention.FIG. 2 is a schematic view of a bioreactor adapted for culturing animal cells according to another embodiment of the present invention.FIG. 3 is a schematic view of a bioreactor adapted for culturing animal cells according to yet another embodiment of the present invention.FIG. 4 is a schematic view of a method for culturing animal cells according to the present invention.

[0019] Several aspects of the present invention are disclosed with reference to flow diagrams and / or block diagrams of methods, devices and systems.

[0020] 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.

[0021] 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

[0022] A description of example embodiments of the invention follows.

[0023] 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 that 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.

[0024] 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.

[0025] 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 nutriment broadly comprises both macronutrients and micronutrients. These encompass essential nutrients like amino acids, vitamins, minerals, proteins, carbohydrates, fats and / or oxygen.

[0026] 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 insidethe vessel working volume. 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.

[0027] 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.

[0028] 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.

[0029] The term “hydrophobic”, in particular “hydrophobic membrane”, can refer, within the meaning of the invention, to a surface such as a membrane that exhibits waterrepelling properties. These surfaces or membranes are specifically designed to allow the passage of gases and vapors while preventing the passage of liquids, particularly water. A hydrophobic membrane will usually exhibit a contact angle greater than 90 degrees.

[0030] The term “rigid” can refer, within the meaning of the invention, to the quality of a material, component, or structure that maintains its shape and does not bend, fold, or deform easily under applied stress or force. This property is characterized by a high resistance to changes in shape or structure when subjected to external pressures, impacts, or mechanical loads. Rigid materials are typically contrasted with flexible or elastic materials, which undergo significant deformation under similar conditions. The rigidity of a material or structure is often quantified in terms of its modulus of elasticity or Young's modulus, with higher values indicating greater rigidity.

[0031] The expression “backbone structure” can refer, within the meaning of the invention to a component, a rigid central framework within a gas exchange device, designed to provide essential structural support and stability. It can be characterized by itssturdy construction and is often the primary load-bearing element. This structure not only supports the physical components of the gas exchange device but also houses and protects critical functional elements like distribution channels or gaseous exchange membrane. Despite its rigidity, the structure is hollow, allowing for efficient passage and distribution of substances necessary for the bioreactor's operation.

[0032] 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.

[0033] The term “a” or “an” as used herein can refer to "one or more" unless explicitly stated otherwise.

[0034] The term "about" as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range.

[0035] The term "substantially" as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.

[0036] The term “significantly” as used herein refers to a measurable and notable change or effect that is beyond normal variations or tolerances expected. This change or effect should be quantifiable, either through empirical data or recognized standards, and must represent an improvement or alteration that is not merely trivial or nominal. The significance of the change or effect can be demonstrated through comparative data, analytical methods, or industry benchmarks that are well-accepted.

[0037] The large-scale cell production such as animal cells for cultivated meat, cultivated leather or large-scale vaccine manufacturing requires a homogeneous distribution of oxygen in a large volume of medium without generating a level of shear stress that could damage the cells.

[0038] Oxygen stands as a crucial nutrient in cell culture. However, its distribution poses problems, particularly in large-scale crops which require very high cell densities. Indeed, oxygen deprivation becomes particularly important at high cell densities as the oxygen consumption is a function of the cell's number. 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 optimizing large-scale cell culture environments.

[0039] The inventors have developed a bioreactor comprising features specifically designed to allow an oxygenation of the culture medium uncorrelated with the level of shear stress, opening the way to large-scale cell culture.

[0040] According to a first aspect, the invention relates to a bioreactor 100 adapted for culturing animal cells. A bioreactor 100 in a system 1 according to the invention is preferably configured to allow cell culture, preferably animal cells such as mammalian, avian or fish cells. In particular, it can be arranged to allow culture of cells in suspension, for example in a fluidized bed.

[0041] A bioreactor 100 according to the invention can be 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.Preferably, the bioreactor 100 is a fluidized bed bioreactor.

[0042] In particular, a bioreactor 100 adapted for culturing animal cells according to the present invention is illustrated in figures 1 to 3.

[0043] A bioreactor 100 according to the present invention comprises: a vessel 110, a gas exchange device 120, a media inlet 130 and a media outlet 135. Preferably, a bioreactor 100 according to the present invention comprises a vessel 110, at least two gas exchange devices 120, and at least one media inlet 130 and at least one media outlet 135.

[0044] As illustrated in the figure 1 , the bioreactor 100 according to the present invention may also comprise: at least one gas inlet 140, at least one gas outlet 145, at least one sampling port and / or at least one harvesting port 180.

[0045] One of the advantages of the present invention is to allow large-scale culture of cells with reasonable costs. Contrary to the knowledge of those skilled in the art, the present invention, preferably, does not include mechanical agitation devices in the vessel 110 of the bioreactor 100 configured to fluidize the cell bed, preferably in the vessel working volume. Advantageously, the bioreactor 100 does not comprise internal and / or external means of mechanical agitation of the culture medium. For example, the bioreactor 100 does not comprise impellers, or stirrers in the vessel working volume.

[0046] As illustrated in Figure 1 , the bioreactor 100 according to the invention comprises a vessel 110. A vessel 110 is usually defining a cultivation liquid volume 111.

[0047] As mentioned, one of the advantages of the present invention is to allow large- scale cell production with reduced waste of resources, including nutrients. Large-scale cell production using the technical solutions of the present invention can be reached through at least two routes.

[0048] In an embodiment, the vessel 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 least1 000 000 liters.

[0049] 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 having a working volume of at least 5000 liters.

[0050] As the system 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 according to the invention can typically comprise more than 10 vessels 110, preferably more than 100 vessels and / or can comprise vessels 110 with working volume of more than 100 liters,preferably with working volume of more than 200 liters.

[0051] 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.

[0052] The vessel 110 can have a diameter of at least 10 centimetres, preferably at least 50 centimetres, more preferably at least 1 meter, even more preferably at least 10 meters.

[0053] In a preferred embodiment, the working volume of the vessel 110 does not comprise impeller or stirrer.

[0054] A bioreactor 100 according to the present invention comprises a gas exchange device 120. Preferably, a bioreactor 100 according to the present invention comprises at least two gas exchange devices 120.

[0055] In a bioreactor according to the invention, a gas exchange device allows a diffusion of dissolved oxygen through a water / air interface where the liquid medium is not fluid dynamically impacted. The gas exchange device 120 is particularly adapted to modify the gas composition of a culture medium circulating in a vessel 110 of a bioreactor 100.

[0056] In particular, a gas exchange device 120 is configured to supply dissolved oxygen through a mechanism, which can be called a passive mechanism, independent of any shear-stress from mechanisms of agitation. Hence, the gas exchange devices 120, protruding in the vessel 110, are configured to supply oxygen through a mechanism that does not modify significantly neither the velocity at the cell-liquid interface nor the viscosity of the media. The gas exchange devices can be configured to supply oxygen in an amount that is decorrelated from the shear-stress applied to the cells in the vessel 110.

[0057] The modification of the gas composition of the culture medium circulating in the vessel 110 of a bioreactor 100 by a gas exchange device 120 is advantageously done by exchange, the exchange taking place by contact with a culture medium within the vessel 110. As mentioned, the gas exchange device 120 is arranged to create an exchange of gas between a fluid within the gas exchange device 120 (usually a gaseous fluid) and thefluid within the vessel 110 (usually a liquid fluid, a culture media). Hence, the gas exchange device 120 does not correspond to a sparger or similar devices configured to only inject gas within a media. There are preferably bidirectional gas flows making it possible, for example, to enrich the culture medium in oxygen and to deplete it in carbon dioxide. Hence, the gas exchange device 120 used in the invention is advantageously arranged to permit and to improve gas exchange between a gaseous fluid within the gas exchange device 120 and a liquid fluid within the vessel 110 of a bioreactor 100.

[0058] Advantageously, a bioreactor 100 according to the invention comprises at least one gas exchange 120 devices. For example, a bioreactor 100 comprises at least two gas exchange devices 120, for example at least five gas exchange devices 120, preferably at least ten gas exchange devices 120, more preferably at least twenty gas exchange devices 120, and even more preferably at least fifty gas exchange devices 120. In particular these gas exchange devices 120 are arranged so that at least part, preferably most of their gaseous exchange surface protrudes in the vessel working volume.

[0059] As it is illustrated in the figure 1, the gas exchange devices 120 can extend from one end of the vessel to the other, effectively spanning its entire length. This setup allows for efficient distribution or collection of fluids across the entire volume of the vessel. The traversing nature of the gas exchange devices 120 ensures that there is a uniform flow distribution, minimizing zones of stagnation or excessive turbulence. This configuration is particularly beneficial as the cell growth necessitates uniform flow distribution. The positioning of the tubes also facilitates easier maintenance and cleaning, as they can be accessed from either end of the bioreactor.

[0060] As it is illustrated in the figure 2, the gas exchange devices 120 can extend from the top of the bioreactor and descend almost to the bottom. The gas exchange devices 120 can make contact with the bottom surface or not. This design is particularly useful in fluidized bed cell culture where the depth of the tubing ensures thorough mixing and aeration. The gas exchange devices 120 can also run from the bottom of the bioreactor upwards. The gas exchange devices 120 can make contact with the top of the vessel or not. This design is particularly useful in fluidized bed cell culture vessels where the depth of the tubing ensures thorough mixing and aeration. By not touching the top or the bottom, this setup avoids interference with any top-mounted or bottom-mounted instruments or inlets (such as media inlet in the bottom, media outlet at the top, sensors, or harvesting or sampling ports at the top or the bottom of the vessel).

[0061] As it is illustrated in the figure 3, the gas exchange devices 120 can be arranged to extend from one side of the bioreactor to the other. This configuration can create a specific flow pattern, influencing the mixing and aeration within the vessel. This arrangement can ensure a more homogenous reaction environment.

[0062] Preferably each gas exchange device 120 has a backbone hollow structure 122. Preferably, a rigid backbone hollow structure 122.

[0063] The backbone hollow structure 122 is advantageously arranged so as not to deform under the action of the flow in the cultivation volume capable of preventing cells from sedimenting. Thus, it should not bend or twist significantly under normal operating conditions of the cell culture system. As it is further described this can be achieved through the use of adequate material; adequate shape and design; and / or adequate anchors.

[0064] The backbone hollow structure 122 is advantageously a rigid structure made of a material or a combination of materials having an Young’s modulus of at least 100 MPa at 20°C. Preferably, the backbone hollow structure 122 is advantageously a rigid structure made of a material or a combination of materials having an Young modulus of at least 120 MPa at 20°C, more preferably at least 150 MPa at 20°C, even more preferably at least 200 MPa at 20°C. For example, the Young’s modulus is preferably measured according to the norm ISO 527:2012 for plastic, and ISO 3312 or ISO 527 for metals. More preferably, the backbone hollow structure 122 has a ring tensile strength of at least 10 MPa, preferably at least 15 MPa, more preferably at least 30 MPa and even more preferably at least 50 MPa.

[0065] The backbone hollow structure 122 may be made of a material or a combination of materials comprising composite material, polymer, ceramics and / or metal. The composite materials are usually formed from two or more distinct materials that retain their identities in the composite mixture. It can for example be a combination of a reinforcement and a matrix. The backbone hollow structure 122 can comprise a composite material made of a polymer reinforced by carbon fibers or glass fibers. The backbone hollow structure 122 can comprise a polymer such as a polytetrafluoroethylene (PTFE), a polyether ether ketone (PEEK), a polypropylene (PP) or their combination. The backbone hollow structure 122 can comprise a ceramic such as alumina or zirconia. The backbone hollow structure 122 can comprise a metal including metals like stainless steel, titanium, or aluminiumalloys. Preferably, when the backbone hollow structure 122 comprises or is made of metal, it is porous and can be made from a sintering process or additive manufacturing.

[0066] This backbone hollow structure 122 may be a cylindrical tube, a rectangular tube, a square tube, an elliptical tube such as an oval tube, a triangular tube, a hexagonal tube or an annular tube. The structure may be straight, coiled or branched but is preferably straight. Preferably, this backbone hollow structure 122 is a cylindrical structure or cylindrical tube.

[0067] The backbone hollow structure 122 is preferably an elongated structure. For example, the backbone hollow structure 122 has an aspect ratio of at least 5. An aspect ratio is defined as the ratio between the external length of the substrate distribution unit 1 and its external diameter. For example, the substrate distribution unit 1 may have an aspect ratio of at least five, preferably at least seven, more preferably an aspect ratio of at least ten and even more preferably at list twenty. In some embodiments, the backbone hollow structure 122 may have an aspect ratio of at least twenty, preferably at least forty, more preferably at least sixty, even more preferably at least eighty.

[0068] The gas exchange device 120 can thus be also elongated. For example, it has a length of at least 10 cm, preferably at least 50 cm, more preferably at least 100 cm, even more at least 200 cm.

[0069] For example, the backbone hollow structure 122 has a cross-sectional area of at least 0.1 cm2. Preferably, the backbone hollow structure 122 has a cross-sectional area of at least 0.25 cm2, more preferably at least 0.5 cm2, even more preferably of at least 1 cm2. For example, the backbone hollow structure 122 has a cross-sectional area of at most 20 cm2. Preferably, the backbone hollow structure 122 has a cross-sectional area of at most 15 cm2, more preferably at most 10 cm2, even more preferably of at most 5 cm2. The cross-sectional area is typically calculated based on the inner dimensions to assess flow capacity and surface area for cell growth. However, it can also be determined from the outer dimensions, which involves accounting for the wall thickness.

[0070] For example, when the backbone hollow structure 122 has a cylindrical structure, the gas exchange device 120 can have an external diameter of at most 5 cm, preferably at most 4 cm, more preferably at most 3 cm and even more preferably at most 2 cm.

[0071] The wall thickness of the backbone hollow structure 122 is of at least 0.1 mm. Preferably, the wall thickness of the backbone hollow structure 122 is of at least 0.2 mm,more preferably at least 0.3 mm, even more preferably of at least 0.5 mm The wall thickness of the backbone hollow structure 122 is of at most 5 mm. Preferably, the wall thickness of the backbone hollow structure 122 is of at most 4 mm, more preferably at most 3 mm, even more preferably of at most 2 mm.

[0072] Typically, the backbone hollow structure 122 can delineate a conduit 123. This conduit 123 is preferably dedicated to the management of the gas flow participating in the gas exchange with the cultivation medium. Hence, the backbone hollow structure 122 is made of a porous material and / or it comprises multiple through-holes or apertures 124, which provide a function as passageways linking the conduit 123 to the exterior of the backbone hollow structure.

[0073] When the backbone hollow structure 122 is made of a porous material, it has a porosity of at least 20 %, preferably at least 30 %, more preferably at least 40 % and even more preferably at least 50%. The porosity of the backbone hollow structure 122 is preferably measured with a porometer and the permeability is measured according to the ISO 4022:2018 standard.

[0074] When the conduit 123 comprises multiple through-holes or apertures 124 this backbone hollow structure 122 can be considered as perforated. The hole pattern can be a uniform or variable. The perforations in the backbone hollow structure 122 can exhibit a diverse range of shapes, including but not limited to cylindrical (round), rectangular, square, elliptical, triangular, hexagonal, or even more complex geometries. Each shape offers unique characteristics in terms of flow dynamics and distribution efficiency. For instance, cylindrical or round holes are standard for uniform flow, while rectangular or square holes might be advantageous for specific directional flows. Elliptical holes can provide a balance between round and rectangular shapes, offering streamlined flow with reduced resistance. Triangular and hexagonal holes could be employed for specific flow patterns or to enhance the structural integrity of the structure, while also influencing the turbulence and mixing efficiency within the bioreactor.

[0075] For example, the holes have an area of at least 0.1 cm2. Preferably, the holes of the perforated backbone hollow structure 122 have an area of at least 0.25 cm2, more preferably at least 0.5 cm2, even more preferably of at least 0.75 cm2. For example, the holes have an area of at most 10 cm2. Preferably, the holes of the perforated backbone hollow structure 122 have an area of at most 7.5 cm2, more preferably at most 5 cm2, even more preferably of at most 2.5 cm2.

[0076] The holes distribution can be a uniform or variable. The ratio of the cumulative surface area of the apertures 124 or holes on the backbone hollow structure 122 to the total surface area of the backbone hollow structure's walls is at least 0.1. Preferably, this ratio is at least 0.2, more preferably at least 0.4, and even more preferably at least 0.5. For example, the ratio can be of at least 0.8 when holes are drilled at high density on the backbone hollow structure's walls.

[0077] Advantageously, the backbone hollow structure 122 is made of a porous material and it comprises multiple through-holes or apertures 124, which provide a function as passageways linking the conduit 123 to the exterior of the backbone hollow structure 122.

[0078] Each gas exchange device 120 has a gaseous exchange surface 121. Preferably, a gas exchange device 120 according to the invention is arranged so that the gaseous exchange surface 121 protrudes in the vessel 110.

[0079] This gas exchange occurs at a gaseous exchange surface 121 level. Each gas exchange device 120 comprises a gaseous exchange surface 121. The gaseous exchange surface 121 is preferably being arranged to allow gas exchange between a liquid phase flowing in the cultivation liquid volume 111 and a gaseous phase flowing in the gas exchange devices 120. The gaseous exchange surface 121 can come from a gaseous exchange membrane which is positioned on the backbone hollow structure. Alternatively, the gaseous exchange surface 121 can be formed by the backbone hollow structure. These embodiments are illustrated in each of the figures 1 to 3 (e.g. left side membrane forming the gas exchange surface and associated with a perforated backbone structure and right side a porous backbone structure forming gas exchange surface).

[0080] For example, the gaseous exchange surface 121 has 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.

[0081] The gaseous exchange surface 121 is preferably hydrophobic. The gaseous exchange surface 121 may exhibit a low surface energy. In particular, the gaseous exchange surface 121 preferably has a surface energy of at most 100 mN / m, preferably 75 mN / m, more preferably 50 mN / m, and even more preferably 25 mN / m.

[0082] In particular, the gas exchange devices 120 can comprise a gaseous exchange membrane positioned on the backbone hollow structure 122 and which form the gaseousexchange surface 121.

[0083] The gaseous exchange membrane can comprise a polymeric material. Preferably, the gaseous exchange membrane comprises polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), or their combination.

[0084] In the vessel 110, the gaseous exchange surface 121 of each of the gas exchange devices 120 can be separated by at least 4 mm from the surface of the other gas exchange devices 120; preferably at least 5 mm, more preferably at least 6 mm, even more preferably at least 7 mm.

[0085] In the vessel 110, the gaseous exchange surface 121 of each of the gas exchange devices 120 can be separated by at most 50 mm from the surface of the other gas exchange devices 120; preferably at most 25 mm, more preferably at most 15 mm, even more preferably at most 10 mm.

[0086] As mentioned, the present invention optimizes the oxygen distribution among the vessel 110. The distribution can be improved by a suitable density of gaseous exchange surfaces 121 within the vessel 110. In particular, the problems of the state of the art can be solved by reaching a cumulated area of gaseous exchange surfaces 121 within a vessel 110 such that the value of the ratio of cumulated surface area of gaseous exchange surfaces 121 within a vessel 110 on the volume of the vessel 110 is of at least 10 cm-1; at least 25 cm-1, at least 50 cm-1, preferably 100 cm-1, more preferably 200 cm-1, even more preferably 300 cm-1, for example even more preferably 500 cm-1.

[0087] A bioreactor 100 according to the present invention comprises at least one media inlet 130 and at least one media outlet 135.

[0088] The media inlet 130 and media outlet 135 can be configured to control the medium flow within the vessel 110. In particular, they are configured to operate a flow in the cultivation volume capable of preventing cells from sedimenting. Also, they can be configured to suspend cells. Preferably, the inlet and outlet are configured to induce, when operating, a media flow between two gaseous exchange surfaces 121.

[0089] The media inlet 130 is generally arranged to allow the introduction of fresh culture medium and / or recycled culture medium into the vessel 110. This can be used to maintain suitable nutrient levels for cell growth. The media inlet 130 is typically designed to ensureaseptic conditions. It may include filters or sterilization mechanisms to prevent contamination. The flow rate of the medium through the media inlet 130 can be controlled, often using pumps and valves. The media inlet 135 may be associated with distribution mechanisms, like nozzles, to evenly distribute the medium within the bioreactor.

[0090] The media outlet 135 can be used for removing culture medium, spent culture medium, and / or waste materials from the bioreactor. In some setups, the media outlet 135 is also used for harvesting the desired biological products.

[0091] The media inlet 130 and the media outlet 135 may include valves and control systems to regulate the flow rate and maintain pressure within the bioreactor.

[0092] The media inlet 130 and outlet 135 may be positioned so that at least part of the gaseous exchange surfaces 121 are positioned between them.

[0093] The media inlet 130 and outlet 135 may be configured to operate simultaneously or sequentially. Preferably, the media inlet 130 and outlet 135 are configured to operate simultaneously and induce within the vessel a continuous flow in order to maintain cells in suspension when applied to a fluidized bed bioreactor.

[0094] While the media inlet 130 is essential for operating the bioreactor, the new features of a bioreactor according to the invention are such that preferably, a ratio between the cumulated area of the media inlet 130 openings to the gaseous exchange surface 121 in the vessel 110 is lower than 1, preferably lower than 0.5, more preferably lower than 0.1, even more preferably lower than 0.01.

[0095] A bioreactor 100 according to the present invention can comprise at least one gas inlet 140. Preferably, a bioreactor 100 according to the present invention can comprise at least one gas inlet 140 and at least one gas outlet 145.

[0096] More preferably, as illustrated in figures 1, 2 and 3, the at least one gas inlet 140 is positioned within the gas exchange device 120.

[0097] The gas inlet 140 and gas outlet 145 can be configured to control the gas flow within the vessel 110. In particular, they can be configured to operate a flow in the cultivation volume capable of preventing cells from sedimenting. Also, they can be configured to suspend cells. As illustrated in the figures 2 and 3, the gas can flow in and out through the same opening. This is particularly suitable when the gas exchange device comprises a sealed end.

[0098] The gas inlet 140 is generally arranged to allow the introduction of fresh gas and / or recycled gas into the vessel 110. This can be used to maintain suitable oxygen levels for cell growth. The gas inlet 140 is typically designed to ensure aseptic conditions. It may include filters or sterilization mechanisms to prevent contamination. The flow rate of the gas through the gas inlet 140 can be controlled, often using pumps and valves.

[0099] As illustrated in figure 1, advantageously, the system can comprise said gas inlet(s) 140 and gas outlet(s) 145 arranged to permit a gas phase flow between them. Preferably said at least one gas inlet 140 is part of the gas exchange device 120. The system can comprise at least two gas inlet(s) 140 with some being arranged in the gas exchange device 120 while one or at least two others are positioned under the cultivation liquid volume to allow bubbling or micro bubbling of gas in the working volume.

[0100] In another aspect, the invention relates to a cell culture system. A cell culture system according to the invention comprises at least one bioreactor 100 according to the invention. Preferably, it comprises at least two bioreactors 100 according to the invention.

[0101] A cell culture system according to the invention can further comprise heatexchange devices (e.g. heating or cooling of the gas phase); a medium container; a gas container; a waste collection media system; a cell collection system.

[0102] A cell culture system according to the invention can comprise a monitoring and adjustment system. Preferably, the monitoring and adjustment system is configured for monitoring and adapting environmental parameters such as pH, temperature, nutrient concentration, and oxygen levels.

[0103] Hence the monitoring and adjustment system can comprise sensors and control devices distributed throughout the cell culture system to continuously monitor environmental parameters like pH, temperature, nutrient concentration, and oxygen levels.

[0104] The collected data can be used to adjust conditions in real-time, ensuring uniformity in nutrient distribution for example.

[0105] A system 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 thus comprises devices configured to regularly monitor cell population using in-line sampling and analytical techniques.

[0106] In particular, the monitoring and adjustment system 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. 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 at an ideal temperature conducive to cell growth. The sensors are placed to continuously monitor the internal temperature of the bioreactor or other components of the system. The monitoring and adjustment system can be programmed to activate heating or cooling mechanisms as necessary to maintain the temperature within a predefined optimal range.

[0107] In particular, the monitoring and adjustment system 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.

[0108] In particular, the monitoring and adjustment system 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 system 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.

[0109] The monitoring and adjustment system 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 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.

[0110] In particular, the monitoring and adjustment system 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 orsalts, 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.

[0111] 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 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 or other components of the system such as the separator.

[0112] As mentioned, all these sensors can be integrated in automated feedback loops configured to automatically adjust conditions within the vessel. Also, the cell culture system 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.

[0113] A cell culture system according to the invention can comprise a harvesting system. Preferably, the harvesting system is adapted for collecting cells, biomass or cellular products from large volumes without compromising quality and eventually asepsis. The harvesting system can be a continuous or semi-continuous harvesting system that allows for the regular collection of cells, biomass or cellular products while maintaining the cell culture’s integrity. The harvesting system can include separation technologies, like membrane filtration or centrifugation, integrated within the cell culture system.

[0114] In another aspect, the invention relates to a method 1000 for culturing animal cells. A method 1000 for culturing animal cells according to the invention preferably comprises the use of a bioreactor 100 according to the invention.

[0115] For example the bioreactor comprises a vessel 110 defining a cultivation volume 111; at least two gas exchange devices 120 protruding in the vessel 110, each gas exchange devices 120 having a gaseous exchange surface 121 in the vessel 110 ; and at least one media inlet 130 and at least one media outlet 135.

[0116] As shown in figure 4, a method according to the invention comprises the following steps:- Adding 1100 culture media through the media inlet 130;Removing 1200 culture media through the media outlet 135;Exchanging gas 1300 at gaseous exchange surface 121 between a gas fluid circulating within the gas exchange devices 120 and the culture media in the cultivation volume.

[0117] Typical gas flow rate can be of at least 0.1 vvm (volume of injected gas per media volume per minute). Also, the method can comprise a configuration of the gas flow rate in order that the flow rate of the gas fluid circulating within the gas exchange devices 120 is of at least 0.1 times the vessel liquid volume per minute.

[0118] During a method according to the invention, the flow rate of the culture media entering the bioreactor 100 is preferably selected to balance the gravitational force exerted on the cells which would cause them to sediment. For example, the flow rate of the culture media entering the bioreactor 100 is calculated in function of the values of the gas flow rate, the gas pressure within the gas exchange device, the gas permeability of the gas exchange membrane, the concentration of oxygen in the gas, the gas exchange surface area 121, and the vessel 110 volume.

[0119] The flow rate of the culture media entering the bioreactor 100 can further be calculated based on the oxygen consumption of the cells in the vessel 110.

[0120] As mentioned, the method induces a certain amount of shear stress due to the fluid flow. However, the technical solution of the present invention can be used to decouple shear stress from oxygenation making both processes independent.Advantageously, to limit the shear stress, at the gaseous exchange surface 121, the superficial velocity of the culture media is of at most 2 cm / hour, preferably at most 1.5 cm / hour, more preferably at most 1.0 cm / hour, even more preferably at most 0.5 cm / hour. Preferably the linear velocity in the vessel 110 is of at least 0.1 cm / hour.

[0121] The fluid flow can be mainly used to balance the gravitational force exerted on the cells which would cause them to sediment. In particular, the media flow rate is configured to induce 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).

[0122] While conventional bioreactor will use agitation or stirring mechanisms to enhance medium homogenization in the vessel 110, the present invention can benefit from a highvelocity culture medium circulation within the vessel 110, to improve culture medium circulation and homogeneity. For example, the medium within the vessel 110 is removed from the vessel 110 at a rate of at least 4 mL / h, more preferably 0.4 L / h, even more preferably 4000 L / h for larger vessels.

[0123] In particular, the flow rate of the media entering the vessel 110 can be of at least 1% of the vessel working volume per hour. Such value can benefit the mass transfer and nutrient availability in the cultivation liquid volume. Preferably, the flow rate of the media entering the vessel 110 can be of at least 2%, more preferably at least 3%, even more preferably at least 4% of the vessel working volume.

[0124] However, in normal culture condition, the flow rate of the media entering the vessel 110 is configured to be at most 20% of the vessel working volume per hour. Preferably, the flow rate of the media entering the vessel 110 is configured to be at most 15% of the vessel working volume per hour.

[0125] In some embodiment, the vessel can further comprise at least one bubbling device allowing bubbling or micro bubbling of gas in the working volume in order to improve the fluidization of the cellular bed.

[0126] The cell culture within the bioreactor 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.

[0127] The harvesting can be done while the bioreactor 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 cells, the culture media comprising the cell biomass can be drained from the bottom of the vessel 110.

[0128] Following the collection of the cell biomass, the cells 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.

[0129] According to another aspect, the present invention can also relate to a biological product obtainable from a method according to the invention. Preferably, the presentinvention can also relate to a cell biomass obtainable from a method according to the invention. More preferably, the present invention can relate to a cell biomass obtained from a method 1000 according to the invention.

[0130] 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 according to the invention does not necessitate the use of shear-stress protectant such as anti-foaming agent.

[0131] The shear-stress protectant agent can preferably be selected among biocompatible non-ionic surfactants or polymers configured to stabilize cell membranes and reduce the shear forces applied to cells, especially in environments with air or gas bubbles. Also, the shear-stress 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.

[0132] In particular, the shear-stress protectant agent is a polyoxyethylenepolyoxypropylene 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.

[0133] 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.

[0134] Preferably an edible food product according to the invention comprises less than 25 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 0.25ppm.

[0135] Advantageously, the edible food product according to the invention can be considered as an ingredient for an alternative to conventional meat products or an as alternative to conventional meat products as such.

[0136] 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.

[0137] 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.

[0138] 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

Claims1. A bioreactor (100) adapted for culturing animal cells, comprising: a vessel (110) defining a cultivation liquid volume (111); at least two gas exchange devices (120) protruding in the vessel (110), each gas exchange devices (120) having a backbone and a gaseous exchange surface (121), said gaseous exchange surface (121) being arranged to control gas exchange between a liquid phase flowing in the cultivation volume (111) and a gaseous phase flowing in the gas exchange devices (120); and at least one media inlet (130) and at least one media outlet (135), said media inlet (130) and outlet (135) being configured to induce a flow in the cultivation volume capable of preventing cells from sedimenting.

2. The bioreactor (100) according to claim 1, wherein the backbone is arranged so as not to deform under the action of the flow in the cultivation volume capable of preventing cells from sedimenting.

3. The bioreactor (100) according to claim 1 or 2, wherein the gas exchange devices(120) protruding in the vessel (110) are configured to supply oxygen in an amount that is decorrelated from the shear-stress applied to the cells in the vessel (110).

4. The bioreactor (100) according to any one of claims 1 to 3, wherein a cumulated area of gaseous exchange surfaces (121) within the vessel (110) is arranged such that a value of a ratio of cumulated surface area of gaseous exchange surfaces(121) within the vessel (110) on the volume of the vessel (110) is of at least 10 cm’ 15. The bioreactor (100) according to any one of claims 1 to 4, wherein the gas exchange devices (120) protruding in the vessel (110) are configured to supply oxygen through a mechanism that do not modify significantly neither the velocity at the cell-liquid interface not the viscosity of the media.

6. The bioreactor (100) according to any one of claims 1 to 5, wherein, in the vessel (110), the gaseous exchange surface (121) of each of the gas exchange devices (120) is separated by at most 50 mm from the surface of the other gas exchange devices (120).

7. The bioreactor (100) according to any one of claims 1 to 6, wherein the gaseous exchange surface (121) has a gas permeability of at least 10 cm3 / m2.d.bar8. A method (1000) for culturing animal cells said method (1000) comprising the use of a bioreactor (100) comprising a vessel (110) defining a cultivation volume (111); at least two gas exchange devices (120) protruding in the vessel (110), each gas exchange devices (120) having a backbone structure and a gaseous exchange surface (121) in the vessel (110); and at least one media inlet (130) and at least one media outlet (135); said method (1000) comprising the following steps: adding (1100) culture media through the media inlet (130); removing (1200) culture media through the media outlet (135); exchanging (1300) gas at gaseous exchange surface (121) between a gas fluid circulating within the gas exchange devices (120) and the culture media in the cultivation volume (111); said media inlet (130) and outlet (135) operating to induce a flow in the cultivation volume capable of preventing cells from sedimenting, said gas exchange devices (120) controlling the gas exchange at the gaseous exchange surface (121) between a liquid phase flowing in the cultivation volume (111) and a gaseous phase flowing in the gas exchange devices (120).

9. The method (1000) according to claim 8, wherein the flow rate of the culture media entering the vessel (110) is selected to balance the gravitational force exerted on the cells which would cause them to sediment, preferably the linear velocity in the vessel (110) is of at least 0.1 cm / hour.

10. The method (1000) according to claim 8, wherein the flow rate of the culture media entering the vessel (110) is of at least 1% of the vessel working volume per hour.

11. The method (100) according to any one of claims 8 to 10, wherein the flow rate of the gas fluid circulating within the gas exchange devices (120) is of at least 0.1 times the vessel liquid volume per minute.

12. The method (100) according to any one of claims 8 to 11, wherein it comprises a monitoring step comprising: monitoring the fluid dynamics within the vessel (110); monitoring the height of the fluidized bed; monitoring the pH levels; monitoring thetemperature; monitoring the cell density; monitoring the nutrient concentration; and / or monitoring the pressure.

13. The method (1000) according to any one of claims 8 to 12, wherein the flow rate of the culture media entering the bioreactor (100) is calculated as a function of the values of the gas flow rate, the gas pressure within the gas exchange device (120), the gas permeability of the gas exchange membrane, the concentration of oxygen in the gas, the gas exchange surface (121) area, and the vessel working volume.

14. A cell biomass obtainable from a method according to any one of claims 8 to 13, said cell biomass comprising less than 50 ppm of shear-stress protectant agent.

15. An edible food product obtainable from cell biomass according to the previous claim, said edible food product comprising less than 45 ppm of shear-stress protectant agent.

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