Gas exchange device for cell culture bioreactor

Abstract

The invention relates to a gas exchange device (1), for cell culture bioreactor (100), having a backbone hollow structure (10), such as a hollow cylindrical structure (10), wherein said backbone hollow structure (10) has an aspect ratio of at least five, and said gas exchange device (1) further comprising a gas exchange membrane (20) supported by the backbone hollow structure (10), the gas exchange membrane (20) having a gas permeability of at least 100 cm³ / m².d.bar.

Description

GAS EXCHANGE DEVICE FOR CELL CULTURE BIOREACTORField 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).[4] In particular, when upscaling animal cell culture, such as mammalian, fish or avian cells, a multitude of interconnected challenges arise, predominantly centered around the management of shear stress, ensuring cost effectiveness, and maintaining operationalintegrity. The enlargement of bioreactor volumes necessitates a mitigation of the impact of altered physical and chemical environments on sensitive animal cells. Among these considerations, the management of shear stress is key. Agitation is necessary to uniform mixing and nutrient distribution in larger volumes, meanwhile it impairs cells by increasing the shear stress cells are exposed to. Yet, the amplification of shear forces can detrimentally affect cell viability and, consequently, the yield and quality of the desired bioproduct.[5] Despite advances in techniques such as low shear horizontal bioreactor designed for the production of animal cells (Celik, Yunus et al. “A novel low shear horizontal bioreactor design for the production of animal cells: Effect of bioreactor dynamics on the 3D spheroid formation of HepG2.” Biochemical Engineering Journal, vol. 196 108952, July 2023), large-scale production of animal cells remains subject to challenges with excessive shear stress, mixing and oxygen transfer.[6] Thus, there is a significant need for an improved approach to animal cell culture.[7] In particular, the upscaling of animal cell-based products manufacturing to industrial levels necessitates transitioning from laboratory-scale bioreactors to much larger systems. The core of these challenges lies in the effective management of larger bioreactor environments, which requires careful conception and calibration at a significantly increased scale. Such an approach should maximize the oxygenation of the cell culture media for large-scale in suspension animal cells production, reduce the environmental footprint of bioprocessing, and lower 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 gas exchange device for cell culture bioreactor, having a backbone hollow structure, such as a hollow cylindrical structure, wherein said backbone hollow structure has an aspect ratio of at least five, and said gas exchange device further comprising a gas exchange membrane supported by the backbone hollow structure, the gas exchange membrane having a gas permeability of at least 10 cm3 / m2.d.bar, preferably of at least 100 cm3 / m2.d.bar.

[0010] A gas exchange device according to the invention allows an improvement of the homogeneous distribution of oxygen in a bioreactor. This improvement is crucial for cell growth and sustainability, as it ensures the 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 at high scale. A key advantage of this invention is its ability to minimize shear stress during the cell culture process. Excessive shear stress can damage delicate cell structures, especially animal cells, impeding growth and productivity. The technology employed in this invention allows for gentle yet effective distribution of the oxygen, ensuring cell viability and integrity are maintained, which is essential for high-quality cell-based product development.

[0011] Moreover, the invention can simplify the maintenance process of bioreactors, which is often a challenging and time-consuming task. This ease of maintenance not only reduces labor and costs but also minimizes the risk of contamination and errors, leading to a more consistent and reliable cell culture process. 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 or any animal cell-based products manufacturing.

[0012] According to other optional features of the gas exchange device according to the invention, it can optionally include one or more of the following characteristics alone or in combination: the backbone hollow structure is arranged so as not to deform under the cell culture bioreactor operation. For example, to prevent cells from sedimenting, a strong while adapted media flow in the cultivation volume is applied, said flow could by its strength constraints the backbone hollow structure and other of the bioreactor’s parts. The backbone hollow structure is therefore further preferablyarranged so as not to move during the operating of the cell culture bioreactor. This can improve the distribution of oxygen in a bioreactor. the backbone hollow structure is made of a material or a combination of materials having a Young’s modulus of at least 100 MPa at 20°C. When the backbone hollow structure is made of a combination of material, at least one has a Young’s modulus of at least 100 MPa at 20°C. This can improve the distribution of oxygen in a bioreactor. the backbone hollow structure is made of a material or a combination of materials comprising composite material, polymer and / or metal. the backbone hollow structure comprises a porous material and it has a porosity of at least 20%. This can improve the distribution of oxygen in a bioreactor. the backbone hollow structure comprise metal and has been manufactured by additive manufacturing or sintering. This can improve the distribution of oxygen in a bioreactor. the backbone hollow structure delineates a conduit, and wherein said conduit presents multiple apertures positioned on its surface. the gas exchange membrane has a pore size of less than 1 pm. This can improve the distribution of oxygen in a bioreactor. the backbone hollow structure has an aspect ratio of at least twenty, preferably at least forty, more preferably at least sixty, even more preferably at least eighty. This can improve the distribution of oxygen in a bioreactor and the scalability. it comprises a sealed end and a gas distribution tube whose opening is positioned at most at 1 cm of a surface of the sealed end. This can improve the distribution of oxygen in a bioreactor. it has an external diameter of at most 5 cm. This can improve the distribution of oxygen in a bioreactor.

[0013] According to another aspect, the invention can also relate to a cell culture bioreactor comprising a gas exchange device according to the invention, for example at least two gas exchange devices according to the invention, preferably at least twenty,more preferably at least fifty, even more preferably at least one hundred.

[0014] According to other optional features of the cell culture bioreactor according to the invention, it can optionally include one or more of the following characteristics alone or in combination: it comprises at least one media inlet at or near the bottom of the vessel, designed to introduce a liquid culture media into the vessel to fluidize the cells; does not comprise any impeller or stirrer.

[0015] According to another aspect, the present invention can also relate to a cell culture system. A cell culture system according to the invention comprises a cell culture bioreactor according to the invention or a gas exchange device according to the invention.

[0016] According to yet another aspect, the present invention relates to a method for cultivating cells in a cell culture bioreactor comprising the use of a gas exchange device according to the invention. In particular, a method for cultivating cells in a cell culture bioreactor according to the invention comprises the steps of: introducing cells to be cultivated and a culture media in a vessel; maintaining the cells in suspension in the vessel through a flow of culture media in the vessel; and oxygenating the culture media through a flow of gas injected in the gas exchange device.

[0017] According to another aspect, the present invention relates to a cell biomass obtainable from a method according to the present invention, said cell biomass comprising less than 50 ppm of shear-stress protectant agent.

[0018] According to yet another aspect, an edible food product obtainable from cell biomass according to the invention, said edible food product comprises less than 45 ppm of shear-stress protectant agent.Brief description of the drawings

[0019] 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 cross-sectional view of a gas exchange device according to thepresent invention.FIG. 2 is a schematic cross-sectional view of a gas exchange device according to another embodiment of the present invention.FIG. 3 is a schematic perspective cross-sectional view of a gas exchange device according to the present invention.FIG. 4 is a schematic perspective cross-sectional view of a gas exchange device according to another embodiment of the present invention.FIG. 5 is a schematic cross-sectional view of a cell culture bioreactor according to the present invention.FIG. 6 is a schematic view of a cell culture system according to the present invention.FIG. 7 is a schematic view of a method for cultivating cells in a cell culture bioreactor according to the present invention.

[0020] Several aspects of the present invention are disclosed with reference to flow diagrams and / or block diagrams of methods, devices and systems.

[0021] 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.

[0022] 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

[0023] A description of example embodiments of the invention follows.

[0024] 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 industrialprocess, 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.

[0025] The terms or expressions “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.

[0026] 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.

[0027] 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.

[0028] 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 andout 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.

[0029] 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.

[0030] The term “hydrophobic”, in particular “hydrophobic membrane”, can refer, within the meaning of the invention, to a membrane that exhibits water-repelling properties. These 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.

[0031] 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.

[0032] 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 its sturdy 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.

[0033] The term "about" as used herein can allow for a degree of variability in a value orrange, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range.

[0034] 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.

[0035] 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.

[0036] The term “a” or “an” as used herein can refer to "one or more" unless explicitly stated otherwise.

[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 optimising large-scale cell culture environments.

[0039] The inventors have developed a new gas exchange device 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 gas exchange device 1 for cell culture bioreactor 100.

[0041] As illustrated figures 1 to 4 gas exchange device 1 for cell culture bioreactor 100 according to the invention preferably comprises a backbone hollow structure 10 and a gas exchange membrane 20.

[0042] A gas exchange device 1 for cell culture bioreactor 100 according to the invention can further comprise a gas inlet 30, a gas distribution tube 31, a sealed end 40, a cavity 50, a removable connector 60, sensors 70 and anchors 80.

[0043] As illustrated in the figures 1 to 4, a gas exchange device 1 for cell culture bioreactor 100 according to the invention can comprise a backbone hollow structure 10, preferably, a rigid backbone hollow structure 122.

[0044] The backbone hollow structure 10 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.

[0045] The backbone hollow structure 10 is advantageously a rigid structure made of a material or a combination of materials having a Young’s modulus of at least 100 MPa at 20°C. Preferably, the backbone hollow structure 10 is advantageously a rigid structure made of a material or a combination of materials having a Young’s 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.

[0046] The backbone hollow structure 10 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 10 can comprise a composite material made of a polymer reinforced by carbon fibers or glass fibers. The backbone hollow structure 10 can comprise a polymer such as a polytetrafluoroethylene (PTFE), a polyether ether ketone (PEEK), a polypropylene (PP) or their combination. The backbone hollow structure 10 can comprise a ceramic such as alumina or zirconia. The backbone hollow structure 10 can comprise a metal including metals like stainless steel, titanium, or aluminium alloys. Preferably, when the backbone hollow structure 10 comprises or is made of metal, it is aporous material and can be made from a sintering process or additive manufacturing.

[0047] This backbone hollow structure 10 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 10 may be straight, coiled or branched but is preferably straight. Preferably, this backbone hollow structure 10 is a cylindrical structure or cylindrical tube.

[0048] The backbone hollow structure 10 is preferably an elongated structure. For example, the backbone hollow structure 10 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 10 may have an aspect ratio of at least twenty, preferably at least forty, more preferably at least sixty, even more preferably at least eighty.

[0049] The gas exchange device 1 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.

[0050] For example, the backbone hollow structure 10 has a cross-sectional area of at least 0.1 cm2. Preferably, the backbone hollow structure 10 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 10 has a cross-sectional area of at most 20 cm2. Preferably, the backbone hollow structure 10 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.

[0051] For example, when the backbone hollow structure 10 has a cylindrical structure, the gas exchange device 1 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. However, in some preferred embodiments, to maximize the gas exchange, the backbone hollow structure has an external diameter of at most 1.5 cm, preferably at most 1 cm, more preferably at most 0.8 cm, even more preferably at most 0.4 cm.

[0052] The wall thickness of the backbone hollow structure 10 is of at least 0.1 mm. Preferably, the wall thickness of the backbone hollow structure 10 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 10 is of at most 5 mm. Preferably, the wall thickness of the backbone hollow structure 10 is of at most 4 mm, more preferably at most 3 mm, even more preferably of at most 2 mm.

[0053] Typically, the backbone hollow structure 10 can delineate a conduit 11. This conduit 11 is preferably dedicated to the management of the gas flow participating in the gas exchange with the cultivation medium. Hence, the backbone hollow structure 10 is made of a porous material and / or it comprises multiple through-holes or apertures 12, which provide a function as passageways linking the conduit 11 to the exterior of the backbone hollow structure.

[0054] When the backbone hollow structure 10 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 10 is preferably measured with a porometer and the permeability is measured according to the ISO 4022:2018 standard.

[0055] When the conduit 11 comprises multiple through-holes or apertures 12 this backbone hollow structure 10 can be considered as perforated. The hole pattern can be a uniform or variable. The perforations in the backbone hollow structure 10 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 110.

[0056] The holes can have an area of at least 0.1 cm2. Preferably, the holes of the perforated backbone hollow structure 10 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. The holes have an area of at most 10 cm2. Preferably, the holes of the perforated backbone hollow structure10 can have an area of at most 7.5 cm2, more preferably at most 5 cm2, even more preferably of at most 2.5 cm2.

[0057] The hole distribution can be a uniform or variable. The ratio of the cumulative surface area of the apertures 12 or holes on the backbone hollow structure 10 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.

[0058] Advantageously, the backbone hollow structure 10 is made of a porous material and it comprises multiple through-holes or apertures 12, which provide a function as passageways linking the conduit 11 to the exterior of the backbone hollow structure 10.

[0059] The gas exchange device 1 further comprises a gas exchange membrane 20. The gas exchange membrane 20 is preferably arranged in the gas exchange device 1 to create a gas permeable barrier between a gas phase inside the gas exchange device 1 and a liquid phase outside the gas exchange device 1.

[0060] The modification of the gas composition in the culture medium circulating in the vessel 110 of a bioreactor 100 by a gas exchange device 1 is advantageously done by an exchange of gases. The gas exchange typically takes place in contact with a culture medium within the vessel 110. As mentioned, the gas exchange device 1 is arranged to create an exchange of gas between a fluid within the gas exchange device 1 (usually a gaseous fluid) and a fluid within the vessel 110 (usually a liquid fluid, a culture medium). Hence, the gas exchange device 1 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 1 according to the invention is advantageously arranged to permit and improve gas exchange between a gaseous fluid within the gas exchange device 1 and a liquid fluid within the vessel 110 of a bioreactor 100.

[0061] The gas exchange device 1 according to the invention has the advantage of introducing a gas into the bioreactor 100 without inducing shear stress in the bioreactor 100. The gas exchange membrane 20 can have 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.

[0062] The gas exchange membrane 20 is preferably hydrophobic such as a hydrophobic gas-permeable membrane. The gas exchange membrane 20 preferably exhibits a low surface energy. In particular, the gas exchange membrane 20 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.

[0063] The gas exchange membrane 20 can comprise polymeric material. Preferably, the gas exchange membrane 20 or hydrophobic gas-permeable wall comprises polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), or their combination. The gas exchange membrane 20 may preferably exhibit high elongation at break, measured in terms of a specific elongation at break percentage, to withstand operational stress. In particular, the gas exchange membrane or the hydrophobic gas-permeable wall preferably can have an elongation at break of at least 100%, preferably 200%, more preferably 300 %, and even more preferably 400%.

[0064] As illustrated in the figure 1, the gas exchange device 1 may further comprise a gas inlet 30.

[0065] The gas inlet 30 can be positioned in the backbone hollow structure 10 for optimal distribution of gas. It typically includes or connects to flow control devices such as valves, pumps, or regulators. These components allow for precise control of the gas flow rate and pressure entering the gas exchange device, which is important for maintaining optimal conditions for cell culture or reactions.

[0066] The gas exchange device 1 may also comprise a gas distribution tube 31. Typically, the gas distribution tube 31 extends from the gas inlet 30 into the gas exchange device 1 to an opening 32 of the gas distribution tube 31. The gas distribution tube 31 can comprise several openings. Preferably, it comprises only one opening which can be positioned opposite to the gas inlet 30.

[0067] As illustrated in the figure 1 to 4, a gas exchange device 1 may comprise a sealed end 40. Typically, the backbone hollow structure 10 in the bioreactor 100 includes the sealed end 40, which is typically located at the bottom of the structure 10.

[0068] The primary function of the sealed end 40 is to provide a boundary for the gaseous fluid within the gas exchange device 1, preventing leakage and ensuring controlled flow within the backbone hollow structure 10. It can also contribute to the overall structural integrity of the backbone hollow structure 10.

[0069] The end is sealed using methods that guarantee airtightness and fluid impermeability. This could involve welding, adhesive bonding, or a fitted cap, depending on the material and design requirements. Alternatively, the sealed end 40 can be part of the backbone hollow structures 10 and / or be a continuation of it.

[0070] The sealed end 40 can be constructed using the same material as the rest of the backbone hollow structure 10 to ensure uniformity in strength, chemical resistance, and compatibility with the bioreactor's operating environment. The gas exchange device 1 comprises a sealed end 40 and a gas distribution tube 31. The sealed end 40 and the gas distribution tube 31 comprise an opening 32. The opening 32 can be positioned at most at 1 cm of the sealed end 40, preferably at most at 0.75 cm, more preferably at most at 0.5 cm, and even more preferably at most at 0.25 cm.

[0071] In addition, a gas exchange device 1 may comprise a cavity 50. Preferably, the walls forming the cavity 50 are not constituted by the gas exchange membrane 20.

[0072] The cavity 50 is preferably positioned near the opening of the gas distribution tube 31. Preferably, the gas distribution tube 31 should be lowered to the bottom of the compartment, preferably touching the bottom, and at the most not higher than 50% of the compartment height. Advantageously, the gas distribution tube 31 comprises an opening within the cavity 50.

[0073] Preferably, a surface of the cavity 50 is positioned at most at 1 cm of an opening 32 of the gas distribution tube 31. Advantageously, the cavity 50 is positioned so that gravity allows precipitation to separate from the membrane 20. Water runs down and is kept out of contact with the gas exchange membrane 20 by staying in the cavity 50 where it is evaporated by the gas inlet 30.

[0074] Moreover, the cavity 50 is preferably positioned near the sealed end 40. More preferably the cavity 50 is constituted at least in part by the sealed end 40. The cavity 50 can be made of a material having a gas permeability of less than 100 cm3 / m2.d.bar.

[0075] A gas exchange device 1 according to the invention may further comprise a removable connector 60.

[0076] A removable connector 60 is preferably configured to permit a removable connection of the gas exchange device 1 within a bioreactor 100. Indeed, the gas exchange device 1, integral to the bioreactor 100 system, is advantageously designed with a focus on facilitating ease of maintenance.

[0077] The removable connector 60 allows for quick detachment and reattachment of the gas exchange device 1 from the main bioreactor 100 body.

[0078] In a preferred embodiment, the removable connector 60 is engineered to ensure aseptic conditions within the bioreactor 100, a secure and leak-proof seal when in operation, especially to maintain aseptic conditions in the main bioreactor 100 body during operation, while also allowing for straightforward disengagement for cleaning, inspection, or replacement. The removable connector 60 includes features such as quick-release couplings or clamps, which enable tool-free removal, enhancing the efficiency of maintenance procedures. In particular, a removable connector 60 can comprise quickrelease couplings, clamps, camlock fittings, valve-integrated connectors, tri-clamp fittings, and / or bayonet fittings.

[0079] Advantageously, the removable connector 60 is disposed on an extremity of the gas exchange device 1.

[0080] A gas exchange device 1 according to the invention may further comprise one or at least two sensors 70.

[0081] Sensors 70 may be configured to measure media quality, oxygen concentration, or both. Sensors 70 may also be configured to interact with processors configured to regulate oxygen diffusion accordingly.

[0082] Sensors 70 can be selected from pH sensors, dissolved oxygen sensors, temperature sensors, conductivity sensors, CO2 sensors, pressure sensors, level sensors, flow sensors, gas analysers, infrared sensors, cell debris and waste sensors, ammonia sensors, lactate sensors, toxins sensors, or salts sensors, and / or humidity sensors.

[0083] A gas exchange device 1 may further comprise anchors 80. Anchors 80 are configured for maintaining the gas exchange device 1 in a fixed position, such as a vertical position or a horizontal position when immersed in a culture media in the vessel 110. Anchors 80 may be selected from clamps, flange connections, and / or mounting arms.

[0084] In another aspect, the invention relates to a cell culture bioreactor 100. A cell culture bioreactor 100 comprises one or at least two gas exchange devices 1 according to the invention.

[0085] A bioreactor 100 in a system 2 according to the invention is preferably configuredto allow cell culture, preferably animal cells, even more preferably avian cells, mammalian cells or fish cells. In particular, it can be arranged to allow culture of cells in suspension, for example in a fluidized bed.

[0086] The bioreactor 100 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. When the system according to the invention comprises at least two bioreactors, the bioreactors can be a combination thereof. Preferably, the bioreactor 100 is a fluidized bed bioreactor.

[0087] As illustrated in the figure 5, the cell culture bioreactor 100 according to the invention can also comprise a vessel 110, a media inlet 135 and a media outlet 130.

[0088] A bioreactor 100 can also comprise sampling port 160 and harvesting port 180.

[0089] As illustrated in Figure 5, the bioreactor 100 according to the invention may comprise a vessel 110.

[0090] As mentioned, one of the advantages of the present invention is to allow large- scale cell production. Large-scale cell production using the technical solutions of the present invention can be reached through at least two routes.

[0091] 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.

[0092] 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 1000 liters, even more preferably at least 2 000. For example, the invention can be suited to vessels having a working volume of at least 5 000 liters.

[0093] 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, morepreferably 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 100 vessels 110 and / or can comprise vessel 110 with working volume of more than 100 liters.

[0094] 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.

[0095] 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.

[0096] In a preferred embodiment, the working volume of vessel 110 does not comprise impeller or stirrer.

[0097] The gas exchange devices 1 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.

[0098] In particular, the gas exchange devices 1 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).

[0099] These oxygenation devices induce minimal shear stress compared to sparging, reducing the risk of cell damage, especially important for sensitive cell lines, such as animal cell lines. Moreover, combined with the control of the medium flow within the vessel 110, such a method 1000 allows a decorrelation between oxygenation and shear stress.

[0100] Typical gas flow rate can be of at least 0.1 vvm (volume of injected gas per media volume per minute).

[0101] Advantageously, the cell culture bioreactor 100 can comprise at least two gas exchange devices 1 , for example at least five, preferably at least ten, more preferably at least twenty, even more preferably at least fifty. In particular these gas exchange devices1 are arranged so that at least part, preferably most of their gaseous exchange surface 12 protrude in the vessel 110 working volume.

[0102] In the vessel 110, the gaseous exchange surface 12 of each of the gas exchange devices 1 may be separated by at least 4 mm from the surface of the other gas exchange devices 1 ; preferably at least 5 mm, more preferably at least 6 mm, even more preferably at least 7 mm.

[0103] In the vessel 110, the gaseous exchange surface 12 of each of the gas exchange devices 1 may be separated by at most 50 mm from the surface of the other gas exchange devices 1 ; preferably at most 25 mm, more preferably at most 15 mm, even more preferably at most 10 mm.

[0104] As mentioned, the present invention optimises the oxygen distribution among the vessel 110. The distribution can be improved by a suitable density of gaseous exchange surfaces 12 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 12 within a vessel 110 such that the value of the ratio of cumulated surface area of gaseous exchange surfaces 12 within a vessel 110 on the volume of the vessel 110 is of 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.

[0105] A cell culture bioreactor 100 comprises a media inlet 135. A media inlet 135 is configured to allow the media to fill the bioreactor 100. A media inlet 135 may be selected from a port, an opening and / or a hole.

[0106] The media inlet 135 may be disposed at or near the bottom of the vessel 110, to introduce a liquid medium into the vessel 110 to fluidize the cells.

[0107] Preferably, the cell culture bioreactor 100 comprises at least one media inlet 135 at or near the bottom of the vessel 110, designed to introduce a liquid medium into the vessel 110 to fluidize the cells.

[0108] In another aspect, the invention relates to a cell culture system 2. A cell culture system 2 according to the invention comprises at least one gas exchange device 1 according to the invention or at least one bioreactor 100 according to the invention, said bioreactor 100 comprising at least one gas exchange device 1.

[0109] As illustrated in the figure 6, a cell culture system 2 according to the invention canfurther comprise distribution transfer lines 200, a separator 300, a fresh media tank 400, a monitoring and adjustment system 500, a separation device 700, a harvesting device 800; and waste media tank 900.

[0110] A cell culture system 2 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.

[0111] As illustrated in Figure 6, the system 2 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 2 is designed to operate under aseptic conditions to avoid contamination, an important aspect in cell culture bioreactors.

[0112] Some of the distribution transfer lines 200 are described hereafter in association with the component they are interacting with.

[0113] As illustrated in Figure 6, a 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 2 involved in recycling.

[0114] In particular, the recyclable media transfer line 210 can be arranged to transport the multiphase flow directly or indirectly to a separator 300. A gas transfer line 280, such as a recycled gas transfer line, can be arranged to transport the gas to be recycled from the separator 300 to the vessel (directly or indirectly).

[0115] As illustrated in Figure 6, a cell culture system 2 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 and / or the nutrient supply unit 1 , waste or waste metabolites level and other gasses such as CO2.

[0116] Hence, the monitoring and adjustment system 500 can comprise sensors andcontrol devices distributed throughout the cell culture system 2 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.

[0117] A system 2 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, nutrient dosing and oxygen dosing. Preferably, the system 2 thus comprises devices configured to regularly monitor cell population using in-line sampling and analytical techniques.

[0118] In particular, the monitoring and adjustment system 500 can be configured to carry out a temperature control system. 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 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.

[0119] 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.

[0120] 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 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.

[0121] The monitoring and adjustment system 500 can be configured to control oxygenlevels 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.

[0122] 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.

[0123] 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 such as the separator 300.

[0124] 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 2 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.

[0125] As illustrated in Figure 6, a system 2 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.

[0126] A system 2 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 710 unit can be used on arecycled 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.

[0127] A system 2 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 centrifugal separators 720 are calibrated to apply a specific centrifugal force, tailored to the density and size of the cells, ensuring efficient and gentle separation.

[0128] As illustrated in Figure 6, a system 2 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.

[0129] The harvesting system 800 can be a continuous or semi-continuous harvesting system that allows for the regular collection of biological products preferably while maintaining the cell culture’s integrity. The harvesting system 800 can include separation technologies, like membrane filtration or centrifugation, integrated within the cell culture system 2.

[0130] 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.

[0131] As described, the cell culture within the bioreactor 100 is closely monitored to determine the optimal time for biological products collection. The biological products are harvested when the cell culture reaches a desired density and viability status.

[0132] 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 or harvesting of the biological products, the culture media comprising the cell biomass can be drained from the bottom of the vessel 110.

[0133] Following the collection, biological products can be subjected to a centrifugation process. This step is designed to separate the cell biomass 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.

[0134] A system 2 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.

[0135] 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.

[0136] 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.

[0137] 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 is based in a high velocity culture medium circulation within the vessel 110, between one or at least two media inlets 135 and one or at least two media outlets 130, to improve culture medium circulation and homogeneity.

[0138] 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. As large-scale applications are accessible with the current invention, the mediumwithin the vessel 110 is removed from the vessel at a rate corresponding to at least 3 % of the volume of the vessel / h, more preferably 4 % of the volume of the vessel / h, even more preferably 5 % of the volume of the vessel / h.

[0139] In another aspect, the invention relates a method 1000 for cultivating cells in a cell culture bioreactor 100 comprising the use of a gas exchange device 1 according to the present invention, in particular the use of at least one gas exchange device 1 according to the present invention.

[0140] An embodiment of a method according to the invention is illustrated in figure 7.

[0141] A method 1000 for cultivating cells comprises the step of: introducing 1100 cells to be cultivated and a culture media in a vessel 110; maintaining 1200 the cells in suspension in the vessel 110 through a flow of culture media in the vessel 110, preferably a controlled flow of culture media in the vessel 110; and oxygenating 1300 the culture media through a flow of gas injected in the gas exchange device 1 , preferably a controlled flow of gas injected in the gas exchange device 1.

[0142] As described, 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.

[0143] 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.

[0144] Following the collection of the biological products such as 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.

[0145] According to another aspect, the present invention can also relate to a biological product obtainable from a method according to the invention. Preferably, the present invention 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 obtainedfrom a method according to the invention.

[0146] 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 gas exchange device 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.

[0147] The shear-stress protectant agent can preferably be selected among biocompatible non-ionic surfactants or polymers configured to stabilize cell membranes to 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] Advantageously, the edible food product according to the invention can be considered as an ingredient for an alternative to conventional meat products or as analternative to conventional meat products as such.

[0152] 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.

[0153] 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.

[0154] 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 gas exchange device (1), for cell culture bioreactor (100), having a backbone hollow structure (10), such as a hollow cylindrical structure (10), wherein said backbone hollow structure (10) has an aspect ratio of at least five, and said gas exchange device (1) further comprising a gas exchange membrane (20) supported by the backbone hollow structure (10), the gas exchange membrane (20) having a gas permeability of at least 100 cm3 / m2.d.bar.

2. The gas exchange device (1) according to claim 1 , wherein the backbone hollow structure (10) is made of a material or a combination of materials having a Young’s modulus of at least 100 MPa at 20°C.

3. The gas exchange device (1) according to claim 1 or 2, wherein the backbone hollow structure (10) comprises a porous material and it has a porosity of at least 20 %.

4. The gas exchange device (1) according to any one of claims 1 to 3, wherein the backbone hollow structure (10) delineates a conduit (11), and wherein said conduit (11) presents multiple apertures (12) positioned on its surface.

5. The gas exchange device (1) according to any one of claims 1 to 4, wherein the gas exchange membrane (20) has a pore size of less than 1 pm.

6. The gas exchange device (1) according to any one of claims 1 to 5, wherein the backbone hollow structure (10) has an aspect ratio of at least twenty, preferably at least forty, more preferably at least sixty, even more preferably at least eighty.

7. The gas exchange device (1) according to any one of claims 1 to 6, wherein it comprises a sealed end (40) and a gas distribution tube (31) whose opening (32) is positioned at most at 1 cm of a surface of the sealed end (40).

8. The gas exchange device (1) according to any one of claims 1 to 7, wherein it has an external diameter of at most 5 cm.

9. A cell culture bioreactor (100) comprising at least two gas exchange devices (1) according to any one of claims 1 to 8.

10. The cell culture bioreactor (100) according to claim 9, comprising at least one media inlet (135) at or near the bottom of the vessel (110), designed to introduce a liquid culture media into the vessel (110) to fluidize the cells.

11. The cell culture bioreactor (100) according to claims 9 or 10, which does not comprise any impeller or stirrer.

12. A cell culture system (2) comprising a cell culture bioreactor (100) according to any one of claims 9 to 11 or a gas exchange device (1) according to any one of claims1 to 8.

13. A method (1000) for cultivating cells in a cell culture bioreactor (100) comprising the use of a gas exchange device (1) according to any one of claims 1 to 8, said method (1000) comprising the steps of: introducing (1100) cells to be cultivated and a culture media in a vessel (110); maintaining (1200) the cells in suspension in the vessel (110) through a flow of culture media in the vessel (110); and oxygenating (1300) the culture media through a flow of gas injected in the gas exchange device (1).

14. A cell biomass obtainable from a method according to claim 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 comprises less than 45 ppm of shear-stress protectant agent.

Images