Nutrient distribution unit for bioreactor

Abstract

The invention relates to a nutrient supply unit (1) for a bioreactor (100) comprising a vessel (110), said nutrient supply unit (1) comprising: a gas exchange device (10), the gas exchange device (10) comprising a gas exchange surface (12) and being configured to introduce a gas into the bioreactor (100), preferably without inducing shear stress in the bioreactor (100); a media distribution device (20), the media distribution device (20) being configured to introduce a culture media into the bioreactor (100); and a media collection device (30), the media collection device (30) being configured to remove the culture media from the bioreactor (100); Said gas exchange device (10), media distribution device (20) and media collection device (30), being arranged to induce a flow of culture media at the gas exchange surface (12).

Description

NUTRIENT DISTRIBUTION UNIT FOR 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 due to poor management of the flow of culture media and heterogeneity in the distribution of nutrients and gas, such as oxygen and carbon dioxide.[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] 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 operational integrity. 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 uniformmixing 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] Moreover, the maintenance of bioreactors during scale-up introduces additional complexities. The task of ensuring consistent environmental conditions, such as oxygen transfer rates and homogenization, becomes increasingly intricate in larger bioreactors. The need for rigorous and ongoing maintenance to uphold operational efficiency and product quality is paramount, underscoring the multifaceted nature of bioreactor scale-up.[6] 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, and maintenance.[7] In particular, the upscaling of animal cell-based products production to industrial levels necessitates transitioning from laboratory-scale bioreactors to much larger systems. Thus, there is a significant need for an improved approach to animal cell culture. Such an approach enhances the mixing and oxygen transfer without increasing the shear stress and enables easier maintenance, 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 nutrient supply unit for a bioreactor comprising a vessel, preferably for cell culture, the nutrient supply unit comprising: o a gas exchange device, the gas exchange device comprising a gas exchange surface and being configured to introduce a gas into the vessel, preferably without inducing shear stress in the vessel; o a media distribution device, the media distribution device being configured to introduce a culture media into the vessel; and o a media collection device, the media collection device being configured to remove the culture media from the vessel; said gas exchange device, media distribution device and media collection device, being arranged to induce a flow of media at the gas exchange surface.

[0010] Such a nutrient supply unit allows an enhancement of the mixing and oxygen transfer in a bioreactor. This improvement is crucial for cell growth and sustainability, as it ensures the cells receive the necessary nutrients and oxygen uniformly. 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, impeding growth and productivity. The technology employed in this invention allows for gentle yet effective mixing, ensuring cell viability and integrity are maintained, which is essential for high-quality cell-based product development. 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.

[0011] According to other optional features of the nutrient supply unit according to the invention, it can optionally include one or more of the following characteristics alone or in combination:it is adapted to protrude within the vessel of the bioreactor. This can improve the mixing and oxygen transfer in a bioreactor.The nutrient supply unit comprises a removable connector on an extremity. This can be particularly relevant when considering the combination of enhancing the mixing and oxygen transfer and enabling easier maintenance.The nutrient supply unit is adapted to protrude within the vessel of the bioreactor. For example, it can protrude at least in part. Preferably, most or all the gas exchange surface protrudes within the vessel of the bioreactor. This can enhance the mixing and oxygen transfer.The gas exchange surface has a gas permeability of at least 10 cm3 / m2.d.bar. This can enhance the mixing and oxygen transfer.The nutrient supply unit has an aspect ratio of at least five. This can enhance the mixing and oxygen transfer.The media distribution device and the media collection device are arranged to create a flow of media to maximizing contact of the culture media with the gas exchange surface. This can enhance the mixing and oxygen transfer.The media distribution device is configured to induce a flow rate of the media entering the vessel to be of at least 1 % of the vessel working volume per hour. This can enhance the mixing and oxygen transfer.The nutrient supply unit further comprises sensors for measuring media quality, oxygen concentration, or both, and for regulating oxygen diffusion accordingly. This can enhance the mixing and oxygen transfer.The nutrient supply unit further comprises anchors for maintaining it in a vertical position when immersed in media. This can be particularly relevant when considering the combination of enhancing the mixing and oxygen transfer and enabling easier maintenance.

[0012] According to another aspect of the present invention, it proposes a bioreactor comprising a vessel and at least one nutrient supply unit according to the invention protruding in the vessel of the bioreactor.

[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 bioreactor is a fluidized bed bioreactor.The bioreactor comprises at least two nutrient supply units.The value of the ratio of cumulated surface area of the gas exchange surface within a vessel on the volume of the vessel is of at least 50 cm-1.- The nutrient supply unit is arranged to allow gas exchange between a gaseous fluid within the gas exchange device, and a liquid fluid within the vessel of the bioreactor.

[0014] According to another aspect of the present invention, the invention relates to a cell culture system comprising at least one nutrient supply unit according to the invention or at least one bioreactor according to the invention.

[0015] According to another aspect of the present invention, the invention relates to a method for cell culture, using a nutrient supply unit comprising a gas exchange device, the gas exchange device comprising a gas exchange surface; a media distribution device; and a media collection device; preferably said nutrient supply unit protruding, at least partially in a vessel of a bioreactor; said method comprising: a step of introducing a gas into the vessel through the gas exchange device; a step of introducing a culture media into the vessel through the media distribution device; and a step of removing the culture media from the vessel through the media collection device.Preferably, the steps of introducing a culture media and removing the culture media induce a flow of culture media at the gas exchange surface. The steps of introducing a culture media and removing the culture media can be done simultaneously.

[0016] This method allows a gentle yet effective mixing, ensuring cell viability and integrity. As a result, the present method 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.

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

[0018] According to yet another aspect of the present invention, the invention relates 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

[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:Figure 1 is a schematic view of a nutrient supply unit according to an embodiment of the invention.Figure 2 is a schematic cross-sectional view of a nutrient supply unit according to an embodiment of the invention.Figure 3 is a schematic view of a bioreactor according to the invention.Figure 4 is a schematic view of a cell culture system according to an embodiment of the invention.Figure 5 is a schematic view of a method of cell culture according to an embodiment of the 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 “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, which can equilibrate the concentration of certain gaseous molecules between two phases. 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 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.

[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 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.

[0034] The term “a” or “an” as used herein can refer to "one or more" unless explicitly stated otherwise.

[0035] The large-scale cell production such as animal cells for cultivated meat, cultivated leather or large-scale vaccine manufacturing requires enhanced mixing and oxygen transfer, reduced shear stress and scalability for industrial production.

[0036] As described below, the inventors have developed a technology that can be used to respond to these problems. Sufficient oxygen and nutrient supply are essential for cell culture, yet their distributions pose challenges in large-scale cultivation. Moreover, the mass transfer characteristics at this scale often result in uneven or insufficient oxygen concentrations within the growth medium. While enhancing agitation in the growth area could facilitate better oxygen diffusion, this approach leads to unwanted shear stress causing cellular damage. Consequently, balancing oxygen supply without compromising cell integrity is a key consideration in optimizing large-scale cell culture environments.

[0037] The inventors have created a nutrient supply unit designed to efficiently distribute the nutrient and, in particular, oxygenate the media at a reduced cost. This innovation, especially suited for fluidized bed bioreactors, paves the way for large-scale cell production that is economically feasible.

[0038] According to the first aspect, the invention relates to a nutrient supply unit 1 for bioreactor 100. In particular, as it is described hereafter, the nutrient supply unit 1 according to the invention is preferably adapted for use in a bioreactor for in suspension culture. Also, the nutrient supply unit 1 according to the invention is preferably adapted for use as a protruding entity, preferably at least partially, in a vessel of a bioreactor.

[0039] As illustrated in figure 1 & 2, a nutrient supply unit 1 for bioreactor 100 according to the invention may comprise a gas exchange device 10, a media distribution device 20 anda media collection device 30.

[0040] In a particularly preferred embodiment of the invention, the gas exchange device 10, media distribution device 20 and media collection device 30 are arranged to induce a flow of media at the gas exchange surface 12. This flow of media can be observed during continuous operation.

[0041] The introduction of media into the vessel 110 can comprise the use of pulses for example, at the onset of the operation. These pulses can vary in duration and intensity. This initial pulsing action is designed to rapidly equilibrate the vessel 110 with the necessary nutrients and conditions required for cell culture. The pulsing nature of the flow ensures that the media is mixed thoroughly, preventing gradients of nutrients, gases, or waste products from developing. This is particularly important in the early stages of cell culture, where cells are adapting to their environment and require a homogenous mixture for optimal growth. However, in standard operational regime, media can be introduced into the cell culture vessel at a constant rate. This ensures a steady supply of nutrients and efficient removal of waste products, creating a stable environment conducive to sustained cell growth.

[0042] A nutrient supply unit 1 for bioreactor 100 according to the invention may also comprise a removable connector 40, sensors 50, anchors 60, and / or a bubbling device.

[0043] The nutrient supply unit 1 may comprise a hollow structure, preferably a hollow cylindrical structure. A hollow structure can refer to a three-dimensional form with at least one internal cavity. In particular, a nutrient supply unit 1 may comprise at least two internal cavities.

[0044] The nutrient supply unit 1 can comprise a structure, such as hollow structure formed by additive manufacturing. Indeed, when the hollow structure corresponds at least to the gas exchange device 10, media distribution device 20 and media collection device 30, it can be advantageously manufactured by additive manufacturing.

[0045] The nutrient supply unit 1 may have an aspect ratio of at least two. An aspect ratio is defined as the ratio between the external length of the nutrient supply unit 1 and its external diameter. For example, the nutrient supply unit 1 may have an aspect ratio of at least five, preferably at least seven and more preferably an aspect ratio of at least ten.

[0046] As illustrated in the figure 1, a nutrient supply unit 1 for bioreactor 100 comprises agas exchange device 10.

[0047] As illustrated in figure 3, a gas exchange device 10 according to the invention can be configured to introduce gas in a vessel 110 of a bioreactor 100. A nutrient supply unit 1 is particularly adapted to modify the gas composition of a culture medium circulating in a vessel 110 of a bioreactor 100.

[0048] The modification of the gas composition in the culture medium circulating in the vessel 110 of a bioreactor 100 by a gas exchange device 10 is advantageously done by exchange, the exchange taking place in contact with a culture medium within the vessel 110. As mentioned, the gas exchange device 10 is arranged to create an exchange of gas between a fluid within the gas exchange device 10 (usually a gaseous fluid) and a fluid within the vessel 110 (usually a liquid fluid, a culture medium). Hence, the gas exchange device 10 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 nutrient supply unit 1 according to the invention is advantageously arranged to permit and improve gas exchange between a gaseous fluid within the nutrient supply unit 1 and a liquid fluid within the vessel 110 of a bioreactor 100.

[0049] The gas exchange device 10 can comprise at least one internal cavity 11 which is arranged to accommodate the gas intended to participate in the gas exchange. The internal cavity 11 of this gas exchange device 10 can be organized, for example in subcavities connected fluidically so as to facilitate and improve the movement of the gas and the bringing into contact of a gas exchange surface 12 with a gas which is renewed over a short time.

[0050] The walls of the internal cavity 11 can be made of a porous material. Preferably, the walls of the internal cavity 11 have a porosity of at least 20%, preferably at least 30%, more preferably at least 40% and even more preferably at least 50%. The porosity is preferably measured with a porometer and the permeability is measured according to the ISO 4022:2018 standard.

[0051] The gas exchange device 10 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 device 10 preferably comprises a gas exchange surface 12.

[0052] The gas exchange surface 12 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.

[0053] The gas exchange surface 12 is preferably hydrophobic. The gas exchange surface 12 can exhibit a low surface energy. In particular, the gas exchange surface 12 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.

[0054] Advantageously, the area of the gas exchange surface 12 represents at least 30% of the total surface area of the nutrient supply unit 1. Preferably, the area of the gas exchange surface 12 represents at least 40% of the total surface area of the nutrient supply unit 1, more preferably at least 50% and even more preferably at least 60%.

[0055] The gas exchange surface 12 can be directly formed by the walls of the internal cavity 11. Alternatively, the cavity 11 may include openings leading to a gas exchange membrane. Hence, advantageously, to allow the gas exchange, the gas exchange device 10 comprises a gas exchange surface 12 that comprises or consists in a gas exchange membrane such as a hydrophobic gas-permeable wall.

[0056] The gas exchange membrane or hydrophobic gas-permeable wall can comprise a polymeric material. Preferably, the gas exchange membrane or hydrophobic gas- permeable wall comprises polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), or their combination.

[0057] The gas exchange membrane or the hydrophobic gas-permeable wall 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%.

[0058] The gas exchange device 10 can comprise a housing 15, for example to maintain the gas exchange surface 12 on the gas exchange device 10 or comprising the gas exchange surface 12. The housing 15, preferably functioning as a structural backbone, is designed to support and align the gas exchange surface 12.

[0059] The housing 15 is advantageously arranged so as not to deform under the actionof 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.

[0060] The housing 15 may be made of a material or a combination of materials comprising composite materials, polymers, ceramics and / or metals. 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 housing 15 can comprise a composite material made of a polymer reinforced by carbon fibers or glass fibers. The housing 15 can comprise a polymer such as a polytetrafluoroethylene (PTFE), a polyether ether ketone (PEEK), a polypropylene (PP) or their combination. The housing 15 can comprise a ceramic such as alumina or zirconia. The housing 15 can comprise a metal including metals like stainless steel, titanium, or aluminum alloys. Preferably, when the housing 15 comprises or is made of metal, it is a porous material and can be made from a sintering process or additive manufacturing.

[0061] When the housing 15 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 housing 15 is preferably measured with a porometer and the permeability is measured according to the ISO 4022:2018 standard.

[0062] The housing 15 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 housing 15 is advantageously a rigid structure made of a material or a combination of materials having an 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. More preferably, the housing 15 have 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.

[0063] The housing 15 can incorporate attachment points for secure gas exchange surface 12 fixation, allowing for even distribution of mechanical stress and facilitating effective gas permeation. The housing 15 can include integrated channels or grooves for fluid flow, enhancing the exposure of the gas exchange surface 12 to the fluids and thereby optimizing the gas exchange process.

[0064] The gas exchange device 10 can also comprise one or at least two fluid channels 13, 14. In particular, the gas exchange device 10 can include channels 13,14, for gaseous fluid flow, situated inside the hollow structure of the gas exchange device 10. These channels 13,14 for example an inlet channel 13 and an outlet channel 14 are arranged to optimize the exposure of the fluids to the gas exchange surface 12, thereby maximizing the efficiency of gas exchange. The gas exchange device 10 can also be coupled with a pressure control system in order to maintain differential pressures across the membrane to enhance gas transfer.

[0065] Preferably, the gas exchange device 10 can be coupled to a temperature control system to maintain optimal conditions for gas solubility and diffusion.

[0066] A nutrient supply unit 1 according to the invention comprises a media distribution device 20.

[0067] The media distribution device 20 may comprise at least one internal cavity 21. The internal cavity 21 can be arranged to accommodate the culture media to be distributed within a vessel 110.

[0068] The media distribution device 20 may comprise a single inlet hole design to distribute media in the vessel 110. However, it preferably comprises a media distribution wall 22. The media distribution wall 22 is configured to be in contact with the culture media of a vessel 110 and to distribute media in the vessel 110.

[0069] The media distribution wall 22 may be made of a material or a combination of materials comprising composite materials, polymers, ceramics and / or metals. 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 distribution wall 22 can comprise a composite material made of a polymer reinforced by carbon fibers or glass fibers. The distribution wall 22 can comprise a polymer such as a polytetrafluoroethylene (PTFE), a polyether ether ketone (PEEK), a polypropylene (PP) or their combination. The distribution wall 22 can comprise a ceramic such as alumina or zirconia. The distribution wall 22 can comprise a metal including metals like stainless steel, titanium, or aluminum alloys. Preferably, when the distribution wall 22 comprises or is made of metal, it is a porous material and can be made from a sintering process or additive manufacturing. For example, the media distribution wall 22 isselected from perforated walls and / or porous surface.

[0070] Preferably the distribution wall 22 is disposed at least partially at one end of the nutrient supply unit 1 to ensure improved media distribution within the vessel 110. In this embodiment, the collecting wall 32 is disposed at least partially at the other end of the nutrient supply unit 1.

[0071] Advantageously, the distribution wall 22 area represents at least 0.1% of the total surface area of the nutrient supply unit 1. Preferably, the surface area of the distribution wall 22 represents at least 0.25% of the total surface area of the nutrient supply unit 1 , more preferably at least 0.5% and even more preferably at least 0.75%.

[0072] However, the distribution wall 22 area can represent at most 10% of the total surface area of the nutrient supply unit 1. Preferably, the surface area of the distribution wall 22 represents at most 7.5% of the total surface area of the nutrient supply unit 1 , more preferably at most 5% and even more preferably at most 2.5%, for example at most 1%.

[0073] The media distribution device 20 can also comprise one or at least two fluid channels 23. In particular, the media distribution device 20 can include channels for liquid fluid flow, situated inside the hollow structure of the media distribution device 20. A channel for example an inlet channel 23 is preferably arranged to optimize the distribution of the culture media in the vessel 110. Preferably, the media distribution device 20 is arranged to inject the media within the vessel towards at least one other distribution device.

[0074] The media distribution device 20 is preferably configured to induce a flow rate of the culture media entering the bioreactor 100 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, and the vessel volume. The flow rate of the culture media entering the bioreactor can further be calculated based on the oxygen consumption of the cells in the vessel.

[0075] The technical solution of the present invention can be used to decouple shearstress from oxygenation making both processes independent. Advantageously, to limit the shear stress, at the gaseous exchange surface, 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.

[0076] 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).

[0077] While conventional bioreactor will use agitation or stirring mechanisms to enhance medium homogenization in the vessel 110, the present invention can benefit from a high velocity 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.

[0078] In particular, the media distribution device 20 is preferably configured to induce a flow rate of the media entering the vessel 110 to 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 media distribution device 20 is preferably configured to induce a flow rate of the media entering the vessel 110 of at least 2%, more preferably at least 3%, even more preferably at least 4% of the vessel working volume.

[0079] However, in normal culture condition, the media distribution device 20 is preferably configured to induce a flow rate of the media entering the vessel 110 of at most 20% of the vessel working volume per hour. Preferably, the media distribution device 20 is preferably configured to induce a flow rate of the media entering the vessel 110 of at most 15% of the vessel working volume per hour.

[0080] The media distribution device 20 can also be coupled with a pressure control system in order to maintain a pressure adapted to allow cell viability and / or cell growth within the vessel 110.

[0081] A nutrient supply unit 1 according to the invention comprises a media collectiondevice 30. A media collection device 30 is preferably configured to recover the culture medium present in the vessel 110 and remove it from the bioreactor.

[0082] The media collection device 30 may comprise at least one internal cavity 31. The internal cavity 31 can be arranged to accommodate the culture media (preferably at least partially spent culture media) to be removed from within the vessel 110.

[0083] The media collection device 30 may comprise a single outlet hole design to recover media from the vessel 110. However, it preferably comprises a media collection wall 32. The media collection wall 32 is configured to be in contact with the culture media of a vessel 110 and to recover the culture media from the vessel 110.

[0084] A media collection wall 32 can share the same features or composition as the media distribution wall 22. In particular the media collection wall 32 is selected from perforated walls and / or porous surface.

[0085] Advantageously, the collection wall 32 area represents at least 0.1% of the total surface area of the nutrient supply unit 1. Preferably, the surface area of the collection wall 32 represents at least 0.25% of the total surface area of the nutrient supply unit 1 , more preferably at least 0.5% and even more preferably at least 0.75 %.

[0086] However, the collection wall 32 area can represent at most 10% of the total surface area of the nutrient supply unit 1. Preferably, the surface area of the collection wall 32 represents at most 7.5% of the total surface area of the nutrient supply unit 1 , more preferably at most 5% and even more preferably at most 2.5%, for example at most 1%.

[0087] Advantageously, the media distribution device 20 and the media collection device 30 are arranged to induce a flow of media at the surface of the gas exchange surface 12. Preferably, the media distribution device 20 and the media collection device 30 are arranged to create a flow of media to maximize contact of the media with the gas exchange surface 12, e.g. the hydrophobic gas-permeable wall, of the gas exchange device 10.

[0088] As illustrated in Figure 2, a nutrient supply unit 1 according to the invention may further comprise a removable connector 40.

[0089] A removable connector 40 is preferably configured to permit a removable connection of the nutrient supply unit 1 within a bioreactor 100. Indeed, the nutrient supply unit 1 , integral to the bioreactor 100 system, is advantageously designed with a focus on facilitating ease of maintenance.

[0090] The removable connector 40 allows for quick detachment and reattachment of at least one nutrient supply unit 1 from the main bioreactor 100 body.

[0091] In a preferred embodiment, the removable connector 40 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 40 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 40 can comprise quickrelease couplings, clamps, camlock fittings, valve-integrated connectors, tri-clamp fittings, and / or bayonet fittings.

[0092] Advantageously, the removable connector 40 is disposed on an extremity of the nutrient supply unit 1.

[0093] As illustrated in figure 2 nutrient supply unit 1 according to the invention may further comprise one or at least two sensors 50.

[0094] Sensors 50 may be configured to measure and / or assess media quality, oxygen concentration, or both. Sensors 50 may also be configured to interact with processors configured to regulate oxygen diffusion accordingly.

[0095] Sensors 50 can be selected from pH sensors, dissolved oxygen sensors, nutrient sensors, temperature sensors, conductivity sensors, CO2 sensors, pressure 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.

[0096] A nutrient supply unit 1 may further comprise at least one anchor 60. Anchors 60 are arranged for maintaining the nutrient supply unit 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 60 may be selected from clamps, flange connections, and / or mounting arms.

[0097] A nutrient supply unit 1 may further comprise at least one bubbling device. Bubbling devices are arranged for bubbling or micro bubbling of gas in the working volume.

[0098] Hence, according to another aspect, the invention relates to bioreactor 100. Advantageously, a bioreactor 100 according to the invention is adapted for growing cells, in particular animal cells, preferably cells in suspension, for example in a fluidized bed.

[0099] A bioreactor 100 according to the invention comprises at least one nutrient supply unit 1 according to the invention. For example, as illustrated in figure 3, a bioreactor 100 according to the invention comprises at least two nutrient supply units 1 according to the invention, preferably at least five, more preferably at least ten, even more preferably at least twenty nutrient supply units 1 according to the invention.

[0100] In a vessel, the gaseous exchange surface of each of the nutrient supply units 1 can be separated by at least 4 mm from the other gaseous exchange surface; preferably at least 5 mm, more preferably at least 6 mm, even more preferably at least 7 mm. In a vessel 110, the gaseous exchange surface of each of the nutrient supply units 1 can be separated by at most 50 mm from the other gaseous exchange surface; preferably at most 25 mm, more preferably at most 15 mm, even more preferably at most 10 mm.

[0101] As mentioned, the present invention optimizes the oxygen distribution among a vessel. 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.

[0102] A bioreactor 100 according to the invention is preferably configured to allow cell culture, preferably animal cells, e.g. avian, fish or mammalian cells. In particular, it can be arranged to allow culture of cells in suspension, for example in a fluidized bed.

[0103] A bioreactor 100 can be selected from: a flow-controlled bioreactor, a airlift bioreactor, a stirred-tank bioreactor, a wave / orbital shaking bioreactor, an airlift bioreactor, fluidised bed bioreactor, a packed bed bioreactor, and / or a hollow-fiber bioreactor. Whenthe cell culture system 2 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.

[0104] A bioreactor 100 comprises a vessel 110. A bioreactor 100 can also comprise sampling ports 160 and harvesting ports 180.

[0105] One of the advantages of the present invention is to allow large-scale culture of cells with reasonable cost. Contrary to the knowledge of those skilled in the art, the present invention, preferably, does not include mechanical agitation means in the vessel 110 of the bioreactor 100. Hence, preferably, the working volume of the vessel 110 does not comprise impellers or stirrers.

[0106] As illustrated in Figure 3, the bioreactor 100 according to the invention comprises a vessel 110. The vessel 110 preferably comprises at least one nutrient supply unit 1 according to the invention that protrudes within it.

[0107] 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.

[0108] In an embodiment, the vessel 110 used for the growth of the cells is very large. Hence, the vessel 110 can have a working volume of at least 100 liters, preferably at least 5 000 liters, more preferably at least 30 000 liters, even more preferably at least 100 000 liters. For example, the invention can be suited to vessels having a working volume of at least 1 000 000 liters.

[0109] 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 vessels 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 5 000 liters.

[0110] As the system according to the invention is adapted for large scale production, it isusually 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 100 vessels 110 and / or can comprise vessel 110 with working volume of more than 100 liters.

[0111] Depending on the configuration, the vessel 110 used in the invention has 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.

[0112] The vessel 110 can have a diameter of at least 10 centimeters, preferably at least 50 centimeters, more preferably at least 1 meter, even more preferably at least 10 meters.

[0113] In a preferred embodiment, the working volume of the vessel 110 does not comprise impellers, or stirrers.

[0114] The vessel 110 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.

[0115] According to another aspect, the invention relates to cell culture system 2. A cell culture system 2 according to the invention comprises at least one nutrient supply unit 1 according to the invention. A cell culture system 2 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. For example, at least 2 bioreactors 100, preferably at least 3, more preferably at least 4.

[0116] A cell culture system 2 according to the invention can further comprise distribution transfer lines 200, a separator 300, a fresh media tank 400, a monitoring and adjustment device 500, a separation device 700, a harvesting device 800, and / or spent media tank 900.

[0117] As illustrated in Figure 4, 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 circulationprocess, 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.

[0118] Some of the distribution transfer lines 200 are described hereafter in association with the component they are interacting with.

[0119] As illustrated in Figure 4, 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.

[0120] 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).

[0121] As illustrated in Figure 4, 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 100 and / or the nutrient supply unit 1, waste or waste metabolites level and other gasses such as CO2.

[0122] Hence, the monitoring and adjustment system 500 can comprise sensors and control 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.

[0123] 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 and nutrient dosing. Preferably, the system 2 thus comprises devices configured to regularly monitor cell population using in-line sampling and analytical techniques.

[0124] 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 2 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.

[0125] 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.

[0126] In particular, the monitoring and adjustment system 500 can be configured to carry out a nutrient level control. Preferably, it comprises sensors designed to measure the concentration of essential nutrients such as glucose, amino acids, vitamins and oxygen 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 trigger the addition of the required nutrients, thereby ensuring that the cells are consistently provided with the necessary components for growth and development.

[0127] The monitoring and adjustment system 500 can be configured to control oxygen levels in the culture medium, ensuring cells receive adequate oxygen for metabolism. Preferably, it comprises dissolved oxygen sensors and / or gaseous oxygen designed to measure the concentration of oxygen in the liquid phase and / or the gas phase.Furthermore, it can be configured to trigger the addition of oxygen when levels fall below a set threshold. In particular, the monitoring and adjustment system 500 can be configured to control the pressure of the gaseous phase providing the oxygen supply. Indeed, the transport of oxygen at the gas exchange surface can scale with the external gas pressure.

[0128] 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 theconcentration 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.

[0129] 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.

[0130] 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.

[0131] As illustrated in Figure 4, 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.

[0132] 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 unit can be used on the recycled media transfer line 220 to ensure that the recycled medium injected in the vessel 110 will not comprise cells. These units 710 incorporate semi-permeable membranes to facilitate the selective passage of the culture medium while retaining cells. A distinguishing feature of these units is the pore size of the membrane, which can be chosen based on the average size of the cells being cultured. This sizing can allow the membrane to prevent the passage of cells, while permitting smaller molecules and waste products to filter through. The membranes are designed for optimal permeability and strength, ensuring minimal impact on the viability of the retained cells.

[0133] A system 2 according to the invention can comprise centrifugal separators 720. These devices 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.

[0134] As illustrated in Figure 4, 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.

[0135] 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.

[0136] The harvesting system 800 can include separation technologies, like membrane filtration or centrifugation, integrated within the cell culture system 2.

[0137] 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 characterized by the type of cell, multiplied by the total reactor volume.

[0138] 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.

[0139] The harvesting can be done while the bioreactor 100 is operating or after that the fluidization has been paused. The cessation of the fluidizing elements allows for the cells to transition from a growth phase to a state that is more conducive to harvesting. For thecollection or harvesting of the biological products the culture media comprising the cell biomass can be drained from the bottom of the vessel 110.

[0140] Following the collection, biological products can be subjected to a centrifugation process. This step is designed to separate the cells from any remaining culture medium and to concentrate the cell biomass. The resulting cell pellet can then be subjected to a washing process using for example a sterile buffer solution. The purpose of this washing process is to remove any impurities or residual media components, further purifying the cell biomass.

[0141] Hence, according to another aspect, the invention relates to method 1000 for cell culture. A method 1000 for cell culture comprises the use of at least one nutrient supply unit 1 according to the invention or at least one bioreactor 100 according to the invention or at least one cell culture system 2 according to the invention.

[0142] In particular, a method 1000 for cell culture can comprise the use of a nutrient supply unit 1 comprising a gas exchange device 10, the gas exchange device 10 comprising a gas exchange membrane 12; a media distribution device 20; and a media collection device 30; said nutrient supply unit 1 protruding in the vessel 110 of the bioreactor 100.

[0143] As illustrated in figure 5, a method according to the invention preferably comprises a step of introducing a gas 1100 into the vessel 110, a step of introducing a culture media 1200 into the vessel 110, and a step of removing the culture media 1300 from the vessel; said steps of introducing a culture media 1200 and removing a culture media 1300 being simultaneous and inducing a flow of culture media at the gas exchange membrane 12 surface.

[0144] 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 obtained from a method according to the invention.

[0145] Preferably, said biological product in particular cell biomass comprising less than50 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, evenmore 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.

[0146] The shear-stress protectant agent can preferably be selected among biocompatible non-ionic surfactants or polymers configured to stabilize cell membranes and 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] Advantageously, the edible food product according to the invention can be considered as an ingredient for an alternative to conventional meat products or as an alternative to conventional meat products as such.

[0151] 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 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.

[0152] 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.

[0153] 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 / or closely and / or inextricably linked to each other, but on the contrary as simple juxtapositions. In addition, the structural and / 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 nutrient supply unit (1) for a bioreactor (100) comprising a vessel (110), the nutrient supply unit (1) comprising: o a gas exchange device (10), the gas exchange device (10) comprising a gas exchange surface (12) and being configured to introduce a gas into the vessel (110), preferably without inducing shear stress in the vessel (110); o a media distribution device (20), the media distribution device (20) being configured to introduce a culture media into the vessel (110); and o a media collection device (30), the media collection device (30) being configured to remove the culture media from the vessel (110); said gas exchange device (10), media distribution device (20) and media collection device (30), being arranged to induce a flow of media at the gas exchange surface (12).

2. The nutrient supply unit (1) according to claim 1, wherein it is adapted to protrude within the vessel (110) of the bioreactor (100).

3. The nutrient supply unit (1) according to any one of claims 1 or 2, wherein the gas exchange surface (12) has a gas permeability of at least 10 cm3 / m2.d.bar.

4. The nutrient supply unit (1) according to any one of claims 1 to 3, wherein it has an aspect ratio of at least five.

5. The nutrient supply unit (1) according to any one of claims 1 to 4, wherein the media distribution device (20) and the media collection device (30) are arranged to create a flow of media to maximize contact of the culture media with the gas exchange surface (12).

6. The nutrient supply unit (1) according to any one of claims 1 to 5, wherein the media distribution device (20) is configured to induce a flow rate of the media entering the vessel (110) to be of at least 1% of the vessel working volume per hour.

7. The nutrient supply unit (1) according to any one of claims 1 to 6, wherein it comprises sensors (50) for measuring media quality, oxygen concentration, or both, and for regulating oxygen diffusion accordingly.

8. A bioreactor (100) comprising a vessel (110) and at least one nutrient supply unit (1) according to any one of claims 1 to 7 protruding in the vessel (110), preferably it is a fluidized bed bioreactor.

9. The bioreactor (100) according to claim 8, wherein it comprises at least two nutrient supply units (1).

10. The bioreactor (100) according to anyone of claims 8 or 9, wherein the value of the ratio of cumulated surface area of gas exchange surface (12) within a vessel (110) on the volume of the vessel (110) is of at least 50 cm-1.

11. The bioreactor (100) according to anyone of claims 8 to 10, wherein the nutrient supply unit (1) is arranged to allow gas exchange between a gaseous fluid within the gas exchange device (10), and a liquid fluid within the vessel (110) of the bioreactor (100).

12. A cell culture system (2) comprising at least one nutrient supply unit (1) according to any one of claims 1 to 7 or at least one bioreactor (100) according to any one of claims 8 to 11.

13. A method (1000) for cell culture, using a nutrient supply unit (1) comprising a gas exchange device (10), the gas exchange device (10) comprising a gas exchange surface (12); a media distribution device (20); and a media collection device (30); said method comprising: a step of introducing a gas (1100) into the vessel (110) through the gas exchange device (10); a step of introducing a culture media (1200) into the vessel (110) through the media distribution device (20); and a step of removing the culture media (1300) from the vessel (110) through the media collection device (30).

14. A cell biomass obtainable from a method according to the previous claim, 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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