Bioreactor for orbitally shaken cell cultures, in particular suspension cultures

Abstract

The invention relates to a bioreactor vessel (1) having a vessel outer wall (2) and a bottom (3), further comprising an integrated inner structure (4) providing at least two additional surfaces (4a), (4b) for the reactor interior space of the vessel, the inner structure (4) being spaced apart from the vessel outer wall (2); and to a method for growing biological cells using the bioreactor vessel.

Description

[0001] The present invention relates to a bioreactor container for oscillating cell cultures, particularly suspension cultures of any type of biological cell, comprising an integrated internal structure providing at least two additional internal surfaces for the reactor space, and a method for growing biological cells using said bioreactor container.

[0002] For gas-consuming organisms such as aerobic cells, growth in suspension cell cultures requires a sufficient supply of oxygen in addition to nutrients; therefore, air is introduced into the liquid culture. Orbital oscillating bioreactors are an important type of bioreactor and typically offer culture volumes ranging from microliters to 2000 liters. These reactors are easy to operate at low operating costs and are therefore a target for improving culture performance. A persistent demand for such bioreactor vessels is to increase the gas input into the cell culture medium.

[0003] For example, US 2010 / 0248995 A1 has considered increasing the air input to the culture medium in an oscillating bioreactor, describing a bioreactor with a specially shaped cross-section. This document shows that a reactor with a cross-section having multiple rounded edges provides a significantly higher air input compared to conventional bioreactors with baffles on the outer surface. The optimal configuration has been shown to be a flower-like cross-section.

[0004] Zhang XW, Stettler M. et al. described a bioreactor for mammalian cells with a volume up to 1000 liters in N Biotechnol 35:68-75 (2008), which includes a helical track attached to the inner surface of the container wall to increase the oxygen supply.

[0005] Zhu L., Song B. and Wang Z. describe a bioreactor consisting of a hollow cylindrical wall in J. Chem Technol Biotechnol 94:2212-2218 (2019), in which cell cultures oscillate in orbit within the hollow wall.

[0006] The purpose of this invention is to provide a bioreactor container for biological cell cultures, which has a configuration that is easy to prepare and effectively increases the gas input into the cell culture.

[0007] This objective is achieved by a bioreactor container as defined in the claims. The bioreactor provided by this invention is effective in the growth of biological cells under specified gaseous conditions.

[0008] According to this specification, "cell culture" refers to a culture of any biological cell, such as cultures of microorganisms (along with suitable host cells) such as bacteria, archaea, algae, fungi (including yeast), viruses / bacteriophages, human or animal cells (such as mammalian, avian, or insect cells), or plant cells. According to the invention, the shaking culture can be a suspension cell culture or an adherent cell culture, with suspension cultures being preferred. In particular, suspension cultures of microorganisms are the focus of this invention.

[0009] According to the present invention, the bioreactor container (1) has an outer wall (2) and a bottom (3), and further includes an integrated internal structure (4) that provides at least two additional surfaces (4a) and (4b) for the internal reactor space of the container, wherein the internal structure (4) is spaced apart from the outer wall (2).

[0010] According to the invention, the bioreactor container (1) may also include more than one internal structure (4), for example, surrounding or side by side with each other, wherein the internal structures may have the same geometry or may be different from each other. If more than one internal structure (4) is integrated into the bioreactor container (1), more than two additional surfaces (4a) and (4b) can be provided. The principles of the invention are explained below with respect to an integrated internal structure (4); however, it should be understood that more than one such internal structure (4) may be placed in the bioreactor container (1), thereby creating, for example, 4, 6, 8 or more additional surfaces.

[0011] It should be understood that the internal structure (4) is placed inside the reactor chamber in such a way that it is within the space surrounded by the outer wall (2) of the container (1) (also referred to herein as the “internal reactor space” or simply the “reactor space”): the liquid contained in the reactor chamber can contact at least two surfaces (4a, 4b) of the internal structure (4).

[0012] The internal structure (4) may include a wall providing an outer surface (4a) and an inner surface (4b), wherein the wall: (i) has at least one opening (5) in at least the region closest to the bottom (3), or (ii) is spaced apart from the bottom (3), or (iii) has at least one opening and is spaced apart from the bottom (3). The term “outer surface” refers to the wall surface facing the outer wall (2) of the container, and the “inner surface” faces the center of the container.

[0013] The walls of the internal structure (4) providing surfaces (4a) and (4b) can have substantially the same or similar geometry as the outer wall (2) of the container, such that the walls providing surfaces (4a) and (4b) extend substantially parallel to the outer wall (2) of the container within the reactor interior space. Thus, for example, if the outer wall (2) of the container is cylindrical, conical, or elliptical, the walls of the internal structure (4) can also preferably be cylindrical, conical, or elliptical. Alternatively, the shapes of the internal structure (4) and the outer wall (2) of the container can be different; for example, they can be independently selected from cylindrical, conical, elliptical, or any other suitable shape. Furthermore, if there is more than one internal structure (4) in the bioreactor container (1), the shapes of the multiple internal structures (4) present in the container (1) can be the same or can be different from each other. Any suitable combination of the shapes of the outer wall (2) of the container and (one or more) internal structures (4) should be considered to fall within the scope of the invention. Preferred shapes are conical, cylindrical, and elliptical, which can be combined arbitrarily. For example, the outer wall (2) of the container (1) may be conical and at least one of the internal structures may be cylindrical or elliptical. In a preferred embodiment, the outer wall (2) of the container (1) may be conical and at least one of the internal structures may be cylindrical, or vice versa. In another preferred embodiment, both the outer wall (2) of the container and at least one internal structure (4) having a wall providing said surfaces (4a) and (4b) are cylindrical.

[0014] Particularly preferred is that the internal structure (4) providing the surfaces (4a) and (4b) is placed inside the reactor space in such a way that the liquid contained in the reactor space in the non-operating state (when not oscillating) can be at least distributed / at least distributed throughout the entire area of ​​the bottom (3) of the container (1), so that the liquid can be present throughout the entire cross-section of the reactor space (depending on the filling height).

[0015] In embodiment (i), in which the internal structure (4) includes a wall having at least one opening (5), it is particularly preferred that the at least one opening (5) (e.g., one, two, three, or four or more openings (5)) is located in the region of the wall closest to the bottom (3). “Region closest to the bottom” is defined as half of the wall in the direction of the bottom (3), preferably one-third of the wall in the direction of the bottom (3), more preferably one-quarter or one-fifth of the wall in the direction of the bottom (3). At least one opening (5) (or, if present, two, three, or four or more openings (5)) may be located at the lower end of the internal structure (the end in contact with the bottom (3)) such that the contact between the internal structure (4) and the bottom (3) is discontinuous, so that liquid present in the container (1) can enter the internal space of the internal structure (4). If one or more openings (5) are located at the lower end of the internal structure (4), the openings (5) preferably have dimensions such that they allow liquid to enter / exit the internal space of the internal structure (4), but retain at least 80% of the contact area between the lower end and bottom (3) of the internal structure (4), preferably at least 85%, more preferably at least 90%, at least 95%, and up to 97% or even up to 98% of the theoretically possible contact area between the lower end and bottom (3) of the internal structure. Thus, the contact area is slightly interrupted by the openings. Examples of non-limiting embodiments of this structure are shown in... Figures 1 to 8 As shown in the image.

[0016] The opening (5) may have any shape selected from circles and semicircles representing circular sectors; ellipses and semi-ellipses representing oval sectors; triangles; rectangles; polygons; wavy shapes or any combination thereof.

[0017] The opening (5) preferably has a size of at least 0.05 mm or at least 0.1 mm (cross-section in at least one direction) in any direction, for example at least 0.2 mm, at least 0.3 mm, at least 0.5 mm, at least 0.7 mm, 0.8 mm, 0.9 mm or at least 1 mm. It should be understood that the size of the opening obviously depends on the overall size of the bioreactor container (1). The larger the container, the larger the size of the internal structure (4) and the opening (5). Therefore, for microreactors typically used at the microliter scale, the opening (5) may be very small, for example 0.1 mm or even smaller, at most a maximum of, for example, 0.5 mm or 0.8 mm. For large reactors providing more than 1000 liters of culture medium, the size of the opening (5) can be up to several centimeters, for example in the range of 0.1 cm to 10 cm, 0.4 cm to 8 cm, 0.8 cm to 5 cm or any other suitable size. It should be understood that if there is more than one opening, not all openings (5) must have the same size / shape / dimension, but the openings can be different from each other (but are not required to).

[0018] Therefore, if necessary, depending on the overall dimensions of the bioreactor container (1), the openings (5) of the internal structure (4) can independently have shapes and sizes that provide cross-sections in the range of 0.1 mm to 10 cm or even lower or higher. The size of the openings (5) is limited only by the requirement to allow liquids and cells contained in the container (1) to enter / exit the internal space of the internal structure (4), but to maintain as much surface area as possible for surfaces (4a) and (4b).

[0019] If there is more than one opening, it is preferable that the openings (5) are unevenly distributed along the circumferential extension of the internal structure (4). If there is more than one opening, it is preferable that two "adjacent" openings are arranged at an angle of 20 to 120°, 25 to 90° or 30 to 60° along the circumference, wherein if there are more than two openings, the angles between the openings need not be the same.

[0020] In embodiment (ii), where the internal structure (4) is spaced apart from the bottom (3), the internal structure (4) does not necessarily have an opening (5), but according to embodiment (iii), it may have an opening. If the internal structure (4) is spaced apart from the bottom (3), the structure (4) may be secured, for example, by a retaining structure (7), one end of which is connected to the internal structure (4), and the other end may be connected to the bottom (3) and / or to the outer wall (2) of the container or any other suitable structure, such as any shell (6). If the internal structure (4) is spaced apart from the bottom (3), it should be understood that “spaced apart” means that there is some free space, such as a narrow slit, between the lower end of the internal structure and the bottom (3). The slit should be narrow enough to allow liquid and growing cells to pass through, however, the liquid is only a very small amount compared to the total amount of liquid present in the bioreactor container (1). Therefore, the slit can be as small as 0.05 mm, and at most 2 mm, for example, in a container (1) with a capacity of more than 1000 liters. It should be understood that the size of the space between the lower end and the bottom (3) of the internal structure (4) obviously depends on the overall size of the bioreactor container (1). The larger the container, the larger the size of the internal structure (4) and the size of the space between the lower end and the bottom (3) of the internal structure (4). Therefore, for microreactors typically used at the microliter scale, this space may be very small, for example 0.05 mm or even smaller, and at most, for example, 0.3 mm. For large reactors provided for more than 1000 liters of culture medium, the space between the lower end and the bottom (3) of the internal structure (4) can have a size of up to several millimeters, for example, in the range of 0.5 to 2 mm or any other suitable size. Therefore, if desired, the space between the lower end and the bottom (3) of the internal structure (4) can have a size in the range of 0.05 mm to 2 mm, or even lower or higher, depending on the overall size of the bioreactor container (1).

[0021] The edges / boundaries of the openings (5) of the internal structure (4) or the edges / boundaries of the lower end of the internal structure (4) are preferably rounded so that there are no sharp ridges or edges where cells growing in the reactor chamber may be damaged or injured.

[0022] According to the invention, the internal structure provides at least two additional surfaces (4a, 4b) for the reactor's internal space. These two additional surfaces can be represented by the outer surface (4a) and inner surface (4b) of the wall of the internal structure (4). The internal structure (4) may also provide additional walls and surfaces, but in a simplified embodiment and for ease of understanding, only one wall of the two surfaces (4a) and (4b) is provided here to explain the invention. Other internal structures (4) providing additional surfaces may enhance the effects described herein.

[0023] Providing at least two additional surfaces inside the reactor chamber allows for increased gas input into the liquid contained in the bioreactor container, as the liquid contacts not only the inner surface (2b) of the outer wall (2) of the container, but also the additional surfaces (4a, 4b), particularly the additional surface 4b. If the liquid contacts a surface, it spreads along that surface due to an adhesion effect. If the bioreactor container is further agitated, preferably orbitally agitated, the liquid bounces on the surface and floats further along it, thereby increasing the liquid surface in contact with the gas present in the bioreactor container. This effect is amplified as the ratio between the surface area provided by surfaces (2b), (4a), and (4b) and the liquid volume increases. It is understood that increasing the contact area between the liquid and gas allows more gas to enter the liquid. Therefore, if the ratio of the total surface area of ​​the internal surfaces of the container (1) (surfaces (2b), (4a), and (4b)) in contact with the liquid to the total liquid volume increases, the bouncing and adhesion / floating effects expand the total contact area between the liquid and gas. Therefore, the additional surface (4b) plays a major role compared to the additional surface (4a), because the inner surfaces (2b) and (4b) are the surfaces along which the liquid flows during operation, i.e., corresponding to the bulk liquid oscillating in the container (1) in the form of "liquid scythes". By providing the additional surface (4b), the number of "liquid scythes" increases, resulting in an increase in the contact area between the surfaces (2b, 4b) of the container (1) and the liquid, allowing the liquid to form a film along each of the surfaces during the container's operating mode (oscillation).

[0024] In a preferred embodiment, the internal structure (4) provides surfaces (4a, 4b), at least a portion of which is parallel to the inner surface (2b) of the outer wall (2) of the container. More preferably, at least one of the surfaces (4a, 4b) is parallel to the inner surface (2b), and more preferably, both are parallel to the inner surface (2b).

[0025] The bioreactor container (1) can have any shape commonly known as a bioreactor container. For example, the outer wall (2) of the container can have a cylindrical, conical, elliptical, oval, triangular, rectangular, polygonal, or any other regular or irregular basic geometric shape (considered as its cross-section). The internal structure (4) can have the same or similar shape as the outer wall (2), wherein the size is reduced, or the shape of the internal structure (4) is different from that of the outer wall (2), but is also one of those mentioned above. Preferably, at least the shape of the internal structure (4) is represented by a cylindrical, conical, or elliptical cross-section. More preferably, both the internal structure (4) and the outer wall (2) of the container have such a cylindrical, conical, or elliptical cross-section. It is highly preferred when both are cylindrical.

[0026] The internal structure (4) is spaced apart from the inner wall (2b) of the container to provide an "outer ring portion of the reactor chamber," which is the space between the inner surface (2b) of the outer wall (2) of the container and the outer surface (4a) of the internal structure (4). Thus, the outer ring portion of the reactor chamber provides two surfaces in contact with the (cell culture) liquid, namely surfaces (2b) and (4a). The spacing between the outer surface (4a) of the internal structure (4) and the inner surface (2b) of the container wall (2) is preferably at least 1 / 15, or at least 1 / 12, or at least 1 / 10 of the total cross-section of the reactor chamber, but preferably no more than 1 / 3 of the total internal space, more preferably no more than 1 / 4, and even more preferably no more than 1 / 5 or no more than 1 / 8. In the case where the bioreactor container (1) has more than one internal structure (4), they can be spaced apart from each other at any distance, preferably at the distance defined above, wherein it may be preferred that each of the internal structures(s) is approximately equidistant from the inner surface (2b) of the “first” internal structure (the internal structure closest to the wall (2)). However, the distance from the outer wall (2) to the center of the container (1) can also vary (increase or decrease).

[0027] In a preferred embodiment, the outer surface (4a) of the internal structure (4) is spaced apart from the inner surface (2b) of the outer wall (2) of the container by a certain distance, such that the cross-section (CS) of the internal structure is... 内 ) and the cross-section of the outer wall of the container (CS) 外 The ratio of the cross-sections is in the range of 0.95 to 0.4, preferably in the range of 0.92 to 0.5, more preferably in the range of 0.9 and 0.55, and most preferably in the range of 0.85 to 0.6, wherein the cross-sections are measured along the bottom (3) in the space between the inner surfaces (2b) and (4b). Particularly preferred is that both the outer wall (2) and the internal structure (4) of the container have a substantially cylindrical shape. For cases where there is more than one internal structure (4) in the container, similar cross-sectional ratios can be assumed, where in this case CS 内 This corresponds to the cross-section of the internal structure closer to the center of the container, while CS 外 This represents the cross-section of the internal structure closer to the outer wall (2).

[0028] In a bioreactor vessel with the dimensions / ratio of the outer wall (2) and internal structure (4) as defined above, the gas exchange conditions inside the vessel are particularly favorable.

[0029] In one embodiment, the internal structure(s) may have an internal cross-section CS of 5 to 1200 mm. 内The size of which depends on the overall dimensions of the bioreactor container (1). It should be understood that the dimensions of the internal structure(4)(s) depend on the dimensions of the bioreactor container (1).

[0030] The bioreactor container of the present invention can be represented, for example, as a single reactor in the form of a container, flask, bottle, tubing, test tube, cup, cell culture plate, or bag, or it can be part of a multi-array having multiple individual containers, such as a multiwell plate, cell culture array, or microtiter plate. The bioreactor container can be a very small reactor container for loading a volume of liquid ranging from 20 μl to 5 ml, but it can also be a small reactor container for loading a volume of liquid greater than 5 ml to about 100 ml, a medium-sized reactor container for loading a volume of liquid greater than 100 ml to about 2 liters, a large reactor container for loading a volume of liquid greater than 2 liters to, for example, 10 liters, or an ultra-large reactor container for loading a volume of liquid greater than 10 liters. Common sizes / dimensions of such containers are well known to those skilled in the art.

[0031] The bioreactor vessel (1), particularly the vessel walls (2) and bottom (3), as well as the internal structure (4) and optional retaining structure (7), can be made of any material commonly known for the preparation of bioreactors, such as any polymeric material, glass, or metal, but is not limited to the aforementioned materials. The vessel walls (2), bottom (3), internal structure (4), and optional retaining structure (7) can be made of the same material or different materials, but are preferably made of the same material. Preferably, the bioreactor vessel, as a whole or in part, is made of glass or a plastic material, such as polystyrene, polyethylene, polypropylene, polyamide, polyether, polyvinyl chloride, polyethersulfone, or polyurethane, but not limited to the aforementioned polymeric materials, or is made of metal. Glass is a preferred material for the bioreactor vessel (1). Glass has a wettable, hydrophilic surface, so if liquid flows along the glass surface during operation (oscillation) of the reactor vessel, a film will form on the surface due to the hydrophilic properties of the glass, which provides suitable high gas exchange conditions. Alternatively, the bioreactor container (1) may be made of any of the aforementioned materials and may be coated with a hydrophilic coating material on its inner surface (e.g., at least on surfaces 2b and 4b).

[0032] Particularly preferred is that at least surfaces (2b), (4a) and (4b) are made of a material that is so hydrophilic that the liquid contained in the container during container oscillation forms a thin film on the surface, resulting in a high degree of gas exchange.

[0033] Bioreactor containers may also include a cover (6). Depending on the type of bioreactor, the cover may be a lid, cover plate, covering membrane, sealing head, fastener, stopper, cap, or any other suitable closure method commonly used to close a bioreactor, wherein preferably, the cover is permeable or allows gas to enter the reactor chamber. For example, microtiter plates, multiwell plates, or petri dishes are typically closed by a lid, membrane, or thin film. Containers, flasks, tubes, or bottles are typically closed by a lid, stopper, cap, or screw closure device.

[0034] In particular, if the cover (6) tightly seals the reactor chamber, a bioreactor container (1) or cover (6) that provides an inlet and / or outlet to allow the addition or removal of any contents of the container or to place any measuring device inside the container may be suitable. Thus, nutrients, gases or liquids can be added to the container via the inlet, and contents of interest, such as growing cells, can be removed via the outlet, even in a continuous manner.

[0035] The bioreactor container of the present invention can be used in any method involving the growth of biological cells, particularly in liquids containing cell cultures, where a high gas input is required into the liquid in contact with the cells. A highly preferred bioreactor is suitable for agitated liquid cell cultures. In this manner, the gas can be air, oxygen, carbon dioxide, nitrogen, a mixture containing at least one of these, or any other desired gas. The gas is preferably an oxygen-containing gas, and more preferably air.

[0036] Therefore, part of the present invention is also a method for growing biological cells under defined gaseous conditions, wherein the bioreactor container (1) as defined in the above disclosure is provided with cells to be grown and is at least partially filled with a liquid culture medium and agitated such that at least the liquid moves in a rotating manner within the container. Preferably, the cells to be grown are aerobic cells; however, the bioreactor container of the present invention can also be used if anaerobic cells are to be grown, provided that a suitable gas is provided within the reactor chamber. The bioreactor container is suitable for suspension cell culture and cell layer culture, but suspension cell culture is particularly preferred.

[0037] To culture the cells, the container (1) is filled with a liquid, such as a culture medium, in an amount such that, in a non-operating state (when the container is not agitated), at least 50% of the orifices (5) of the internal structure (4), preferably at least 75%, even more preferably at least 90%, and most preferably all of the orifices, are immersed in the liquid. Even more preferably, not only are the orifices immersed in the liquid, but the degree to which the liquid contacts the surfaces (4a) and (4b) is such that the liquid flows along said surfaces during rotation, thereby increasing the total surface area of ​​the liquid.

[0038] Attached image:

[0039] exist Figure 1 In the diagram, the bioreactor container (1) is shown in a tilted top view. The bioreactor container (1) includes an outer wall (2) having an outer surface (2a) and an inner surface (2b), a bottom (3), and an internal structure (4) having an outer surface (4a) and an inner surface (4b), as well as three circumferentially distributed openings (5) at the lower end of the internal structure (4). The bioreactor container shown has no covering (6).

[0040] Figure 2 It shows the relationship with Figure 1 In similar implementation schemes provided, the opening is not Figure 1 Instead of a triangle, it is square, and the internal structure includes only two openings. Furthermore, the figure illustrates how the cross-section CS of the container (1) is defined. 外 and CS 内 Outer cross-section CS 外 Corresponding to the total cross-section defined by the inner surface (2b) of the outer wall (2) of the container, the inner cross-section CS 内 This corresponds to the cross-section defined by the inner surface (4b) of the internal structure (4).

[0041] Figure 3 The bioreactor container (1) is shown in a tilted top view, in which the internal structure (4) is spaced apart from the bottom (3) by a retaining structure (7).

[0042] Figure 4 A cross-sectional view of the bioreactor container is shown, which includes an outer wall (2) and an internal structure (4) having two openings (5) near the bottom. The outer wall (2) and the internal structure (4) of the bioreactor container have different geometries (the outer wall is a “top-open” conical shape, and the internal structure is a cylindrical shape).

[0043] Figure 5 A cross-sectional view of a bioreactor container is shown, comprising an outer wall (2) and an internal structure (4) having an opening (5) near the bottom. The outer wall (2) and the internal structure (4) of the bioreactor container have the same geometry, both being cylindrical; however, the internal structure (4) is shorter than the outer wall (2).

[0044] Figure 6A cross-sectional view of the bioreactor container is shown, comprising an outer wall (2) and an internal structure (4) having two openings (5) near the bottom. The outer wall (2) and the internal structure (4) of the bioreactor container have the same geometry, both being cylindrical, and have the same height.

[0045] Figure 7 A cross-sectional view of a bioreactor container is shown, comprising an outer wall (2) and an internal structure (4) having an opening (5) near the bottom. The outer wall (2) and the internal structure (4) of the bioreactor container have different geometries (the outer wall is cylindrical and the internal structure is a “top-open” conical shape).

[0046] Figure 8 A bioreactor container is shown in an oblique side view. The bioreactor container includes an outer wall (2) and two internal structures (4), each having an opening near the bottom (3). The outer wall (2) and the internal structures (4) of the bioreactor container have the same geometry.

[0047] Figure 9 The illustration shows the oxygen transfer rate (capacity) entering different volumes of liquid compared to a conventional bioreactor container, the liquid being oscillated in a bioreactor container according to the invention (see Examples 1 and 2).

[0048] Figure 10 The illustration shows improved oxygen transfer into a liquid that is agitated in a bioreactor vessel according to the invention (see Examples 1 and 2).

[0049] Figure 11 The illustration shows the oxygen transfer rate (volume) entering a liquid that oscillates at different rotational speeds in a bioreactor vessel according to the invention (see Examples 1 and 3). Example

[0050] Example 1: Experimental Design:

[0051] Three types of cylindrical bioreactor containers with the following dimensions were prepared using glass:

[0052]

[0053] IS = Internal structure (cylindrical)

[0054] *Four openings, each a square, cut at the bottom of the internal structure.

[0055] Bioreactor containers A, B, and C have each been filled with the same liquid volume (V) as defined below.L The oxygen transfer rate was determined by sulfite oxidation, as detailed in the paper "Optical method for the determination of the oxygen-transfer capacity of small bioreactors based on sulfite oxidation" published by Hermann R. et al. on September 5, 2001 in Biotechnology and Bioengineering, Vol 74, No. 5 (John Wiley & Sons, Inc.).

[0056] Experimental setup:

[0057] 0.5M sulfite oxidation method, and detection of color change:

[0058] Oscillator: Kühner Lab Shaker LSR-V-12.5, oscillation diameter d0 = 25mm

[0059] Sulfite system: 0.5M NaSO3 (Bernd Kraft, Duisburg, Germany) in 12mM degassed phosphate buffer (derived from 0.5M Na2HPO4·2H2O and 0.5M NaH2PO4·2H2O (Roth, Karlsruhe, Germany), 2.4*10 -5 M-bromothymol blue (Sigma-Aldrich, Steinheim, Germany), adjusted to pH 8 or 10 with 30% H₂SO₄ (w / w) (Bernd Kraft, Duisburg, Germany). -7 M CoSO4·7H2O (Sigma-Aldrich, SteinheimGermany)

[0060] Aseptic sealing device: Thomson Ultra Yield FlaskAirOTop Enhanced Seal for Ultra Yield 2.5L Flask (Thomson Instrument Company, Oceanside, USA)

[0061] camera: The Samsung Galaxy A3 (2016) features the "IntervalCam" application.

[0062]

[0063]

[0064] t ox = The time until the reaction ends (color change)

[0065] v O2 = Stoichiometric coefficient of oxygen (0.5)

[0066] c SO3 =Amount of sulfite used

[0067] In the charts showing the results of embodiments of the present invention ( Figures 9 to 11 In the text, the internal structure is referred to as a "tube".

[0068] OTR max Maximum oxygen transfer rate (capacity)

[0069] Example 2: Based on the filling volume V L Determine OTR max

[0070] In the first approach, the transfer of oxygen into the liquid is considered based on the fill volume of a bioreactor vessel with the dimensions described above. Generally, oxygen transfer capacity decreases as volume increases because oxygen transfer primarily occurs at the liquid surface in contact with the gas. If the liquid is supplied in a vessel of the same size (provided the entire bottom of the vessel is covered), the smaller the liquid volume, the greater the ratio of liquid surface area to liquid volume. Here, we consider how vessel design affects oxygen transfer capacity.

[0071] The following samples have been tested (using container designs A, B, and C respectively).

[0072] 1.V L =5ml, shake at 250rpm;

[0073] 2.V L =10ml, shake at 250rpm;

[0074] 3.V L =15ml, shake at 250rpm;

[0075] 4.V L =10ml, shake at 300rpm;

[0076] 5.V L =15ml, shake at 300rpm.

[0077] Figure 9 It shows two rotational speeds ( Figure 9 A: 250rpm Figure 9 B: Results of oxygen transfer capacity dependent on container fill volume at 300 rpm. It can be seen that the oxygen transfer rate increases due to the presence of the internal structure (“tubes”), where the narrow gap between the outer wall of the container and the internal structure improves the OTR. max It is significantly better than the OTR with a wider gap. max Larger. Increasing the rotational speed will significantly increase the oxygen transfer rate (will Figure 9 A and Figure 9 B is compared).

[0078] Figure 10 This demonstrates the degree of improvement in oxygen transfer that can be achieved by incorporating an internal structure (tubes): in Figure 10 As can be clearly seen in Figure A, at 250 rpm, the improvement in oxygen transfer achievable at higher volumes is much stronger, both in wide-gap and narrow-gap vessels. At 300 rpm, the improvement is almost independent of the fill volume, see... Figure 10 B. Both figures show that the improvement obtained by using a smaller distance between the outer wall and the internal structure (narrow gap) is significantly greater than the improvement obtained by using a larger distance (wide gap). However, both designs have a significant positive impact on oxygen transfer compared to bioreactors without any internal structure.

[0079] Example 3: Determining OTR based on rotational speed n max

[0080] Furthermore, the transfer of oxygen into the liquid has been considered based on the rotational speed (n) of the bioreactor vessel having the aforementioned dimensions. It is generally known that oxygen transfer capacity increases with increasing rotational speed because the liquid bounces and flows more along the inner wall surface of the vessel, thus increasing the liquid surface area by forming a film on the inner surface of the wall. Since oxygen transfer primarily occurs at the liquid surface in contact with the gas, gas exchange increases with the increase of this surface area.

[0081] Figure 11 A shows the improvement in oxygen transfer observed in samples 2 and 4. Figure 11 B illustrates the improved oxygen transfer in samples 3 and 5. It can be seen that, at both rotational speeds, the provision of the internal structure providing additional surface area increases oxygen transfer into the liquid. The stronger effect of the narrower gap can be explained by providing a larger area of ​​additional internal surface (the internal surface of the internal structure) to form a liquid film.

[0082] The dimensions and design of the bioreactor vessels shown in the above embodiments should be considered illustrative. Specific dimensions defined herein may vary. In practice, bioreactor vessels of larger or smaller dimensions can be fabricated; however, it is particularly preferred that the proportions of the dimensions, especially the proportions of the walls, space, cross-section, and openings, substantially correspond to the dimensions described herein.

Claims

1. A method for growing biological cells under specified gaseous conditions, wherein a bioreactor container (1) is provided with cells for growth and at least partially filled with liquid, and the bioreactor container is agitated such that at least the liquid moves in a rotational manner inside the container, the bioreactor container (1) having an outer wall (2) and a bottom (3), and further comprising at least one integrated internal structure (4) providing at least two additional surfaces (4a), (4b) for the internal reactor space of the container, the internal structure (4) being spaced apart from the outer wall (2), wherein the internal structure (4) includes walls providing an outer surface (4a) and an inner surface (4b), wherein the walls (i) It has at least one opening (5) in at least the area closest to the bottom (3), or (ii) spaced apart from the bottom (3), or (iii) Having one or more openings and spaced apart from the bottom (3); in, Providing the at least two additional surfaces (4a) and (4b) inside the reactor chamber allows for increased gas input into the liquid contained within the bioreactor vessel; and The one or more openings (5) therein are located at least at the lower end of the inner structure (4) that contacts the bottom (3) of the container, and the openings (5) have dimensions such that at least 80% of the contact area between the lower end of the inner structure (4) and the bottom (3) is retained, and up to 98% of the theoretically possible contact area between the lower end of the inner structure and the bottom (3).

2. The method according to claim 1, wherein the dimensions are such that at least 80% of the contact area between the lower end and the bottom (3) of the internal structure (4) is retained, and up to 97% of the theoretically possible contact area between the lower end and the bottom (3) of the internal structure.

3. The method according to claim 1, wherein the dimensions are such that at least 85% of the contact area between the lower end and the bottom (3) of the internal structure (4) is retained, and up to 98% of the theoretically possible contact area between the lower end and the bottom (3) of the internal structure.

4. The method according to claim 1, wherein the dimensions are such that at least 85% of the contact area between the lower end and the bottom (3) of the internal structure (4) is retained, and up to 97% of the theoretically possible contact area between the lower end and the bottom (3) of the internal structure.

5. The method according to claim 1, wherein the dimensions are such that at least 90% of the contact area between the lower end and the bottom (3) of the internal structure (4) is retained, and up to 98% of the theoretically possible contact area between the lower end and the bottom (3) of the internal structure.

6. The method according to claim 1, wherein the dimensions are such that at least 90% of the contact area between the lower end and the bottom (3) of the internal structure (4) is retained, and up to 97% of the theoretically possible contact area between the lower end and the bottom (3) of the internal structure.

7. The method according to claim 1, wherein the dimensions are such that at least 95% of the contact area between the lower end and the bottom (3) of the internal structure (4) is retained, and up to 98% of the theoretically possible contact area between the lower end and the bottom (3) of the internal structure.

8. The method according to claim 1, wherein the dimensions are such that at least 95% of the contact area between the lower end and the bottom (3) of the internal structure (4) is retained, and up to 97% of the theoretically possible contact area between the lower end and the bottom (3) of the internal structure.

9. The method of claim 1, wherein, depending on the overall size of the bioreactor container, the one or more openings (5) and / or the space from the bottom (3) have a size of at least 0.05 mm in any direction.

10. The method of claim 9, wherein the one or more openings (5) and / or the space from the bottom (3) have a size in any direction ranging from 0.1 mm to 10 cm.

11. The method according to any one of claims 1 to 10, wherein the one or more openings (5) may have any shape selected from: a circle or a semicircle representing a circular sector; an ellipse or a semi-ellipse representing an oval sector; a polygon; a wavy shape or any combination thereof.

12. The method of claim 11, wherein the polygon is a triangle or a rectangle.

13. The method according to any one of claims 1 to 10, wherein the outer wall (2) of the container and the internal structure (4) have the same or different geometries, which are independently selected from cylindrical, conical or elliptical shapes.

14. The method according to claim 13, wherein both the outer wall (2) of the container and the internal structure (4) are cylindrical.

15. The method according to any one of claims 1 to 10, comprising more than one of the internal structures (4) such that more than two additional surfaces are provided within the container (1).

16. The method of claim 15, wherein at least four additional surfaces are provided within the container (1).

17. The method of claim 15, wherein at least six additional surfaces are provided within the container (1).

18. The method of claim 15, wherein at least eight additional surfaces are provided within the container (1).

19. The method according to any one of claims 1 to 10, wherein the interval between the outer surface (4a) of the internal structure (4) and the inner surface (2b) of the container wall (2) is at least 1 / 15 of the cross-section of the total reactor chamber, but not more than 1 / 3 of the cross-section of the total internal space.

20. The method according to claim 19, wherein the interval between the outer surface (4a) of the internal structure (4) and the inner surface (2b) of the container wall (2) is at least 1 / 12 of the cross-section of the total reactor chamber, but not more than 1 / 4 of the cross-section of the total internal space.

21. The method according to claim 19, wherein the interval between the outer surface (4a) of the internal structure (4) and the inner surface (2b) of the container wall (2) is at least 1 / 10 of the cross-section of the total reactor chamber, but not more than 1 / 5 of the cross-section of the total internal space.

22. The method according to claim 19, wherein the interval between the outer surface (4a) of the internal structure (4) and the inner surface (2b) of the container wall (2) is at least 1 / 10 of the cross-section of the total reactor chamber, but not more than 1 / 8 of the cross-section of the total internal space.

23. The method according to any one of claims 1 to 10, wherein the outer surface (4a) of the internal structure (4) is spaced apart from the inner surface (2b) of the outer wall (2) of the container by a certain distance, such that the cross-section CS of the internal structure is... 内 The cross-section CS of the outer wall of the container 外 The ratio is in the range of 0.95 to 0.4, wherein the cross sections are measured along the bottom (3) in the space between the inner surfaces (2b) and (4b).

24. The method of claim 23, wherein the ratio is in the range of 0.92 to 0.

5.

25. The method of claim 23, wherein the ratio is in the range of 0.9 and 0.

55.

26. The method of claim 23, wherein the ratio is in the range of 0.85 to 0.

6.

27. The method of claim 15, wherein each of the internal structures (4) is spaced apart from each other or has a cross-section CS for the outer wall (2) of the container and the internal structure (4) as defined in claims 23 to 26. 内 With cross section CS 外 The ratio.

28. The method according to any one of claims 1 to 10, wherein the bioreactor is a single reactor in the form of a container, or part of a multi-array having a plurality of individual containers.

29. The method of claim 28, wherein the container is a flask.

30. The method of claim 28, wherein the container is a bottle.

31. The method of claim 28, wherein the container is a pipe.

32. The method of claim 28, wherein the container is a tube.

33. The method of claim 28, wherein the container is a cup.

34. The method of claim 28, wherein the container is a bag.

35. The method according to any one of claims 1 to 10, wherein the bioreactor container further comprises a cover (6).

36. The method according to any one of claims 1 to 10, further comprising an inlet and / or an outlet, allowing the addition or removal of any contents of the container or the placement of any measuring device inside the container.

37. Use of a bioreactor container as defined in any of the preceding claims for growing biological cells.

38. The use according to claim 37, wherein the growing biological cells are grown under aerobic conditions.

39. The use according to claim 37 or 38, wherein the biological cell is selected from microorganisms such as bacteria, fungi, yeast, algae or archaea, or animal cells, human cells or plant cells.

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