Bioreactors for orbital shaking of cell cultures, especially suspension cultures.

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to a bioreactor vessel for shaking cell culture, particularly suspension culture, of any biological cell type, comprising an integrated internal structure that provides at least two additional inner surfaces in the reactor space, and a method for growing biological cells using the bioreactor vessel. [Background technology]

[0002] As gas consumption increases (e.g., aerobic biological cells in suspension cell culture), in addition to providing nutrients, sufficient supply of oxygen and other gases, i.e., injection of air into the liquid medium, becomes necessary. Orbital shaking bioreactors are an important type of bioreactor and are commonly available in culture volumes ranging from the μL range to 2,000 L. Such reactors are easy to operate with low operating costs and are therefore targeted for improving culture performance. A continuing demand for such bioreactor vessels is the ability to increase the amount of gas injected into the cell culture medium.

[0003] Increasing the amount of air injected into the culture medium of a shaking bioreactor has been studied, for example, in Patent Document 1, which describes a bioreactor with a uniquely formed cross-section. This document shows that a reactor with a cross-section having multiple curved edges clearly allows for a greater amount of air injection than commonly used bioreactors with baffles on the outer surface. The best mode is shown to be a cross-section that resembles a flower.

[0004] In Non-Patent Document 1, Zhang XW, Stettler M, et al. describe a bioreactor for mammalian cells with a maximum volume of 1000 L, in which a helical track is attached to the inner surface of the container wall to increase the oxygen supply.

[0005] In Non-Patent Document 2, Zhu L., Song B., and Wang Z. describe a bioreactor having a hollow cylinder wall in which cell cultures are orbitally shaken within the hollow wall. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] U.S. Patent Application Publication No. 2010 / 0248995 [Non-patent literature]

[0007] [Non-Patent Document 1] N Biotechnol 35: 68-75 (2008) [Non-Patent Document 2] J. Chem Technol Biotechnol 94: 2212-2218 (2019) [Overview of the project] [Problems that the invention aims to solve]

[0008] The object of the present invention was to provide a bioreactor vessel for biological cell cultures that is easy to prepare and has settings that are effective in increasing the amount of gas injected into the cell culture. [Means for solving the problem]

[0009] This objective is achieved by the bioreactor vessel defined in the claims. The bioreactor provided by the present invention is effective in a method for growing living cells under specified gas conditions.

[0010] According to this specification, “cell culture” means a culture of any living cell (e.g., bacteria, archaea, algae, fungi (including yeast), microorganisms such as virus / phage cultures (with suitable host cells), human or animal cells such as mammals, bird or insect cells, or plant cells). According to the present invention, a shaking culture may be a suspension cell culture or an adherent cell culture, with suspension cultures being preferred. The present invention focuses particularly on suspension cultures of microorganisms.

[0011] According to the present invention, the bioreactor container (1) has an outer container wall (2) and a bottom (3), and further has an integrated internal structure (4) that provides at least two additional surfaces (4a) and (4b) to the internal space of the container. The internal structure (4) is spaced apart from the outer container wall (2).

[0012] According to the present invention, the bioreactor vessel (1) may also have a plurality of internal structures (4) that surround or are arranged side by side. These internal structures may have the same geometric shape or may be different from one another. When a plurality of internal structures (4) are incorporated into the bioreactor vessel (1), more than two additional surfaces (4a) and (4b) may be provided. The principle of the present invention will be described below with reference to a single integrated internal structure (4), but it should be understood that a plurality of such internal structures (4) can be arranged within the bioreactor vessel (1), which may result in, for example, four, six, eight or more additional surfaces.

[0013] It should be understood that the internal structure (4) is positioned inside the reactor container, i.e., inside the space enclosed by the outer wall (2) of the container (1) (also called the "internal reactor space" or simply the "reactor space"), such that the liquid placed in the reactor container can come into contact with at least two surfaces (4a, 4b) of the internal structure (4).

[0014] The internal structure (4) may comprise walls that provide an outer surface (4a) and an inner surface (4b). The walls may (i) have at least one opening (5) in the region closest to the bottom (3), or (ii) be spaced apart from the bottom (3), or (iii) have at least one opening and be spaced apart from the bottom (3). The term "outer surface" refers to the surface of the wall facing the outer wall (2) of the container, and "inner surface" refers to the surface facing the center of the container.

[0015] The walls of the internal structure (4) that provide the surfaces (4a) and (4b) can essentially take the same or similar geometric shape as the outer wall (2) of the container, so that the walls providing the surfaces (4a) and (4b) extend essentially parallel to the outer wall (2) of the container inside the internal reactor space. Therefore, for example, if the outer wall (2) of the container is cylindrical, conical, or elliptical, it is preferable that the walls of the internal structure (4) are also cylindrical, conical, or elliptical. Alternatively, the shapes of the internal structure (4) and the outer wall (2) may be different. For example, they may be independently selected from cylindrical, conical, elliptical, or other suitable shapes. And, if there are multiple internal structures (4) in the bioreactor container (1), the shapes of the internal structures (4) present in the container (1) may be the same or different from each other. Any suitable combination of shapes of the outer wall (2) and the internal structure (4) shall be considered to be included in the provisions of the present invention. Preferred shapes are conical, cylindrical, and elliptical, and these may be combined in any combination. For example, the outer wall (2) of the container (1) may be conical and the internal structure (at least one of them) may be cylindrical or elliptical, or the outer wall (2) of the container (1) may be cylindrical and the internal structure (at least one of them) may be conical or elliptical. In one preferred embodiment, the outer wall (2) of the container (1) may be conical and the internal structure (at least one of them) 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 walls providing surfaces (4a) and (4b) are cylindrical.

[0016] The internal structure (4) providing the surfaces (4a) and (4b) is preferably positioned inside the reactor space such that, in a non-operating (non-shaking) state, the liquid placed in the reactor space can be dispersed over at least the entire area of ​​the bottom (3) of the container (1), and as a result, the liquid can be present over the entire cross-section of the reactor space (depending on the filling height).

[0017] In embodiment (i) where the internal structure (4) comprises a wall having at least one opening (5), it is particularly preferred that at least one opening (5) (for example, one, two, three or four, or more) is arranged in the region of the wall closest to the bottom (3). The "region closest to the bottom" is represented by half in the direction of the bottom (3) of the wall, preferably one-third of the wall, more preferably one-fourth or one-fifth in the direction of the bottom (3) of the wall. At least one opening (5) (or, if present, two, three or four, or more openings (5)) can be arranged at the lower end of the internal structure (the end in contact with the bottom (3)). In this case, the contact portion between the internal structure (4) and the bottom (3) is not continuous so that the liquid in the container can enter the internal space of the internal structure (4). When the opening (5) is arranged at the lower end of the internal structure (4), the opening (5) allows the inflow of liquid into the internal space of the internal structure (4) / the outflow of liquid from the internal space of the internal structure (4), but preferably at least 80% of the contact region between the lower end of the internal structure (4) and the bottom (3), more preferably at least 85%, even more preferably at least 90%, at least 95%, up to 97%, and even up to 98% of the theoretically possible contact region between the lower end of the internal structure and the bottom (3) remains. Thus, the contact region is slightly interrupted by the opening. Examples of non-limiting embodiments of such a structure are shown in FIGS. 1-8.

[0018] The opening (5) can have any shape selected from a circle, a semi-circle representing a sector, an ellipse, a semi-ellipse representing an oval, a triangle, a rectangle, a polygon, a wave shape, or a combination thereof.

[0019] The opening (5) preferably has a dimension (cross-section in at least one direction) of at least 0.05 mm or at least 0.1 mm 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 dimension of the opening is undoubtedly influenced by the overall dimensions of the bioreactor vessel (1). The larger the vessel, the larger the dimensions of the internal structure (4) and the opening (5). Thus, in the case of microreactors commonly used on the μL scale, the opening (5) can be very small, for example, from 0.1 mm or less up to a maximum of 0.5 mm or 0.8 mm. In the case of large reactors provided for culture media exceeding 1000 L, the opening (5) can have dimensions of 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 dimensions. If there are multiple openings, it should be understood that not all openings (5) need to have the same size / shape / dimension, and the openings may be different from each other (although this is not necessary).

[0020] Thus, depending on the overall dimensions of the bioreactor vessel (1), the opening (5) of the internal structure (4) may, if necessary, independently have a shape and dimension that provides a cross-section in the range of 0.1 mm to 10 cm, or less or more than that. The dimension of the opening (5) enables the liquid and cells contained in the vessel (1) to enter and exit the internal space of the internal structure (4), and is only limited by the requirement of keeping the surface areas of the surfaces (4a) and (4b) as large as possible.

[0021] If there are multiple openings, the openings (5) are preferably distributed non-uniformly along the circumferential direction of the internal structure (4). If there are multiple openings, it is preferable that two "adjacent" openings are provided along the circumferential direction at an angle of 20 degrees to 120 degrees, 25 degrees to 90 degrees, or 30 degrees to 60 degrees. If there are three or more openings, this angle does not need to be the same between the openings.

[0022] 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 may have one depending on embodiment (iii). When the internal structure (4) is spaced apart from the bottom (3), the structure (4) may be held in place by, for example, a retaining structure (7). The retaining structure (7) is attached at one end to the internal structure (4) and at the other end to the bottom (3) and / or the outer wall (2) of the container, or to any other suitable structure, such as any closure (6). When the internal structure (4) is spaced apart from the bottom (3), “spaced apart” should be understood to mean that there is free space, such as a narrow slot, between the lower end of the internal structure and the bottom (3). The slot must be narrow enough for liquid and growing cells to pass through, but the amount of liquid is very small compared to the total amount of liquid present in the bioreactor container (1). Therefore, the slots can be reduced to approximately 0.05 mm to a maximum of 2 mm in containers with a capacity exceeding 1000 L. It should be understood that the dimensions of the space between the lower end (4) of the internal structure and the bottom (3) are undoubtedly dependent on the overall dimensions of the bioreactor container (1). The larger the container, the larger the dimensions of the internal structure (4) and the dimensions of the space between the lower end (4) of the internal structure and the bottom (3). Therefore, in the case of microreactors commonly used on a μL scale, the space can be very small, for example, 0.05 mm or less to a maximum of, for example, 0.3 mm. In the case of large reactors provided for culture media exceeding 1000 liters, the space between the lower end (4) of the internal structure and the bottom (3) can have dimensions of several centimeters at most, for example, in the range of 0.5 to 2 mm, or any other appropriate dimension. Therefore, depending on the overall dimensions of the bioreactor container (1), the space between the lower end of the internal structure (4) and the bottom (3) may be in the range of 0.05 mm to 2 mm, or less or more, as needed.

[0023] The edges / boundaries of the opening (5) or the lower edges / boundaries of the internal structure (4) are preferably rounded so as not to have any sharp protrusions or rims that could damage or injure cells growing in the reactor vessel.

[0024] According to the present invention, the internal structure provides at least two additional surfaces (4a, 4b) to the internal reactor space. These two additional surfaces may 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 further walls and surfaces, but for the sake of ease of understanding in a simple embodiment, the present invention will be described herein by reference to one wall that provides two surfaces (4a) and (4b). Further internal structures (4) that provide additional surfaces may enhance the effects described.

[0025] By providing at least two additional surfaces on the inside of the reactor container, the amount of gas injected into the liquid contained in the bioreactor container can be increased. This is because the liquid not only comes into contact with the inner surface (2b) of the outer wall (2) of the container, but also with the additional surfaces (4a, 4b), particularly additional surface 4b. When the liquid comes into contact with a surface, it spreads along that surface due to the adhesion effect. Furthermore, when the bioreactor container is shaken (preferably orbital shaking), the liquid bounces off the surface and flows further along that surface. This increases the liquid surface area that comes into contact with the gas present in the bioreactor container. This effect improves as the ratio of the surface area of ​​surfaces (2b), (4a), and (4b) to the liquid volume increases. It can be seen that increasing the contact area between the liquid and gas allows more gas to be introduced into the liquid. Therefore, as the total surface area of ​​the inner surfaces of container (1) that come into contact with the liquid (surfaces (2b), (4a), and (4b)) increases relative to the total liquid volume, the total contact area between the liquid and the gas increases due to bounce and adhesion / buoyancy effects. Thus, the inner surfaces (2b) and (4b) are the surfaces along which the liquid flows during operation (i.e., during shaking of container (1) in the form of a "liquid sickle"), and since they correspond to the bulk liquid, the added surface (4b) plays a more significant role than the added surface (4a). By providing the added surface (4b), the number of liquid sickles increases, and as a result, the contact area between the surfaces (2b, 4b) of container (1) and the liquid increases, allowing the liquid to form a film along each surface during the operating mode (shaking) of the container.

[0026] In a preferred embodiment, the internal structure (4) provides surfaces (4a, 4b) on which at least a portion is parallel to the inner surface (2b) of the outer wall (2) of the container. More preferably, at least one, preferably both, of the surfaces (4a), (4b) is parallel to the inner surface (2b).

[0027] The bioreactor vessel (1) may have any commonly known bioreactor vessel shape. For example, the outer wall (2) of the vessel may be cylindrical, conical, elliptical, egg-shaped, triangular, rectangular, polygonal, or other geometric, regular or irregular (considered cross-sectional) basic shapes. The internal structure (4) may have the same or similar shape as the outer wall (2), but with reduced dimensions. Alternatively, the shape of the internal structure (4) may be different from the outer wall (2), but one of the aforementioned shapes. At least the shape of the internal structure (4) is preferably represented by a cylindrical, conical, or elliptical cross-section, more preferably both the internal structure (4) and the outer wall (2) have such cylindrical, conical, or elliptical cross-sections, and very preferably both are cylindrical.

[0028] The internal structure (4) is spaced apart from the inner surface (2b) of the container wall and provides the “outer portion of the reactor container.” This is the space between the inner surface (2b) of the outer container wall (2) and the outer surface (4a) of the internal structure (4). Thus, this outer portion of the reactor container provides two surfaces (surfaces (2b) and (4a)) that come into contact with the (cell culture) medium. The outer surface (4a) of the internal structure (4) is preferably spaced apart from the inner surface (2b) of the container wall (2) by at least 1 / 15, or at least 1 / 12, or at least 1 / 10 of the cross-section of the entire reactor container, provided that it is preferably 1 / 3 or less, more preferably 1 / 4 or less, and even more preferably 1 / 5 or 1 / 8 or less of the cross-section of the entire internal space. If the bioreactor container (1) has multiple internal structures (4), they can be spaced apart from each other by any distance, preferably by the previously defined distance, and it is preferable that the internal structures are approximately the same distance from each other as the distance from the "first" internal structure (the internal structure closest to the wall (2)) to the inner surface (2b). However, this distance may vary, increase, or decrease from the outer wall (2) toward the center of the container (1).

[0029] In a preferred embodiment, the outer surface (4a) of the internal structure (4) is the cross section (CS) of the outer wall of the container. out Cross-section of the internal structure relative to (CS) inThe ratio of the internal structure (4) to the outer wall (2) of the container 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 to 0.55, and most preferably in the range of 0.85 to 0.6, and this cross section is measured along the bottom (3) of the space between the inner surfaces (2b) and (4b). It is particularly preferable that both the outer wall (2) of the container and the internal structure (4) have an essentially cylindrical shape. For multiple internal structures (4) present in the container, it can be assumed that the ratio of the cross section is similar, and in this case the CS in This corresponds to the cross-section of the internal structure located near the center of the container, while CS out This represents a cross-section of the internal structure located near the outer wall (2) of the container.

[0030] In a bioreactor vessel having the outer wall (2) and internal structure (4) of the dimensions / ratio defined above, the gas exchange conditions inside the vessel are particularly advantageous.

[0031] In one embodiment, the internal structure has an internal cross-section CS of 5 to 1200 mm. in It may have this size, and this size depends on the overall dimensions of the bioreactor container (1). Please understand that the dimensions of the internal structure (4) depend on the dimensions of the bioreactor container (1).

[0032] The bioreactor vessel of the present invention can be represented by a single reactor in the form of, for example, a container, flask, bottle, pipe, tube, cup, cell culture plate, or bag, or it can be part of a multi-array (e.g., a multi-well plate, cell culture array, or microtiter plate) comprising multiple individual vessels. The bioreactor vessel may be a very small reactor vessel to which a volume of liquid in the range of 20 μL to 5 mL should be loaded, but it may also be a small reactor vessel to which a volume of liquid in the range of more than 5 mL but not exceeding about 100 mL should be loaded, a medium reactor vessel to which a volume of liquid in the range of more than 100 mL but not exceeding about 2 L should be loaded, a large reactor vessel to which a volume of liquid in the range of more than 2 L but not exceeding, for example, 10 L should be loaded, or a very large reactor vessel to which a volume of liquid exceeding 10 L should be loaded. The general sizes / dimensions of such vessels are well known to those skilled in the art.

[0033] The bioreactor vessel (1), particularly the vessel walls (2) and bottom (3), as well as the internal structure (4) and any retaining structure (7), can be made from any material commonly known to be used in the construction of bioreactors (such as any polymer material, glass, or metal), and are not limited to those mentioned. The vessel walls (2), bottom (3), and internal structure (4), as well as any retaining structure (7), may be made from the same or different materials, but are preferably made from the same material. Preferably, the bioreactor vessel is made, in whole or in part, from glass, or a plastic material (e.g., polystyrene, polyethylene, polypropylene, polyamide, polyether, polyvinyl chloride, polyethersulfone, or polyurethane, and not limited to the polymer materials mentioned), or metal. Glass is a preferred material for the bioreactor vessel (1). Because glass has a hydrophilic surface that wets easily, when liquid flows along the glass surface during the operation of the reactor vessel (shaking), the hydrophilicity of the glass causes the liquid to form a film on the glass surface, and this film provides suitable high gas exchange conditions. Alternatively, the bioreactor container (1) can be made from any of the aforementioned materials and may be coated on its inner surface (for example, at least surfaces 2b and 4b) with a hydrophilic coating material.

[0034] It is particularly preferable that at least surfaces (2b), (4a), and (4b) be made from a highly hydrophilic material such that the liquid contained in the container forms a thin film on its surface during shaking, thereby achieving a high gas exchange rate.

[0035] The bioreactor vessel may further have a cover (6). Depending on the type of bioreactor, the cover may be a lid, cover plate, cover film, closure head, fastener, plug, cover cap, or any other suitable closure means commonly used to close the bioreactor. The cover is preferably gas permeable or allows gas to enter the reactor vessel. For example, microtiter plates, multiwell plates, or petri dishes are often closed with cover plates, films, or membranes. Containers, flasks, tubes, or bottles are generally closed with lids, plugs, caps, or screw fasteners.

[0036] In particular, if the cover (6) securely closes the reactor vessel, it may be appropriate to provide an inlet and / or outlet on the bioreactor vessel (1) or closure (6) to allow for the addition or removal of any contents of the vessel, or the placement of any measuring devices inside the vessel. Thus, nutrients, gases, or liquids can be added to the vessel through the inlet, and the contents of interest (e.g., growing cells) can be continuously discharged through the outlet.

[0037] The bioreactor vessel of the present invention can be used in any method including the proliferation of biological cells, particularly in liquids containing cell cultures, and requires a high gas injection rate into the liquid in contact with the cells. Very preferably, the bioreactor may be used in shaking liquid cell cultures. The gas may be air, oxygen, carbon dioxide, nitrogen, a mixture containing at least one of them, or any other desired gas. The gas is preferably an oxygen-containing gas, and more preferably, the gas is air.

[0038] Therefore, part of the present invention is also a method for growing biological cells under specified gas conditions. The bioreactor vessel (1) defined in the above disclosure comprises cells to be grown, is at least partially filled with liquid culture medium, and is shaken so that at least the liquid rotates within the vessel. The cells to be grown are preferably aerobic cells, but if anaerobic cells must be grown, the bioreactor vessel of the present invention can also be used, provided that the appropriate gas is supplied into the reactor vessel. The bioreactor vessel is suitable for suspension cell culture and cell layer culture, but suspension cell culture is particularly preferred.

[0039] For culturing cells, the container (1) is filled with a liquid, such as culture medium, in an inactive state (the container is not being shaken), such that at least 50%, preferably at least 75%, more preferably at least 90%, and most preferably all of the orifice (opening) (5) of the internal structure (4) is submerged in the liquid. It is even more preferable to not only submerge the opening in the liquid, but also to bring the liquid into contact with the surfaces (4a) and (4b) to such an extent that the liquid flows along the surfaces (4a) and (4b) during rotation, thereby increasing the total surface area of ​​the liquid. [Brief explanation of the drawing]

[0040] [Figure 1] Figure 1 shows a view of a bioreactor container (1) from an oblique angle above, including a container outer wall (2) having an outer surface (2a) and an inner surface (2b), a bottom (3), an internal structure (4) having an outer surface (4a) and an inner surface (4b), and three openings (5) distributed circumferentially at the lower end of the internal structure (4). The bioreactor container is shown without a cover (6). [Figure 2]Figure 2 shows an embodiment similar to that of Figure 1. The opening is square rather than triangular as in Figure 1, and the internal structure has only two openings. Furthermore, this figure shows how the cross-sections CSout and CSin of the container (1) are defined. The outer cross-section CSout corresponds to the entire cross-section defined by the inner surface (2b) of the outer wall (2) of the container, and the inner cross-section CSin corresponds to the cross-section defined by the inner sum surface (4b) of the internal structure (4). [Figure 3] Figure 3 shows a view of the bioreactor container (1) from an oblique angle above. The internal structure (4) is separated from the bottom (3) by the holding structure (7). [Figure 4] Figure 4 shows a cross-sectional view of a bioreactor container having an outer wall (2) and an internal structure (4) with two openings (5) near the bottom. The outer wall (2) and internal structure (4) of the bioreactor container have different geometric shapes (the outer wall is a cone with an open top, and the internal structure is cylindrical). [Figure 5] Figure 5 shows a cross-sectional view of a bioreactor container having an outer wall (2) and an internal structure (4) with one opening (5) near the bottom. The outer wall (2) and internal structure (4) of the bioreactor container have the same geometric shape and are both cylindrical, but the internal structure (4) is shorter than the outer wall (2). [Figure 6] Figure 6 shows a cross-sectional view of a bioreactor container having an outer wall (2) and an internal structure (4) with two openings (5) near the bottom. The outer wall (2) and internal structure (4) of the bioreactor container have the same geometric shape, both are cylindrical, and are of the same height. [Figure 7] Figure 7 shows a cross-sectional view of a bioreactor container having an outer wall (2) and an internal structure (4) with one opening (5) near the bottom. The outer wall (2) and internal structure (4) of the bioreactor container have different geometric shapes (the outer wall is cylindrical, and the internal structure is a cone with an "open top"). [Figure 8]Figure 8 shows a bioreactor container viewed from an oblique angle, which has an outer wall (2) and two internal structures (4) each having one opening near the bottom (3). The outer wall (2) and internal structures (4) of the bioreactor container all have the same geometric shape. [Figure 9] Figure 9 shows a graph of the oxygen transfer rate (volume) to liquids of different volumes shaken in the bioreactor container according to the present invention, compared to a general bioreactor container (see Experimental Examples 1 and 2). [Figure 10] Figure 10 shows a graph illustrating the improvement in oxygen transfer to the liquid shaken in the bioreactor container according to the present invention (see Experimental Examples 1 and 2). [Figure 11] Figure 11 shows a graph of the oxygen transfer rate (volume) to liquids shaken at different rotational speeds in a bioreactor container according to the present invention (see Experimental Examples 1 and 3). [Examples]

[0041] Experimental Example 1: Experimental Design Three types of cylindrical bioreactor containers were prepared from glass of the following dimensions.

[0042] [Table 1]

[0043] Bioreactor containers A, B, and C are each defined as having the same liquid volume (V) LThe samples were filled with ) and shaken under aerobic (air) conditions at 21°C. The oxygen transfer rate was measured using the sulfite oxidation method, which was detailed in the article "Optical method for the determination of the oxygen-transfer capacity of small bioreactors based on sulfite oxidation" by Hermann R. et al., published in Biotechnology and Bioengineering, Vol. 74, No. 5, September 5, 2001 (John Wiley & Sons, Inc).

[0044] Experimental setup 0.5M sulfite oxidation method for detecting color changes: - Shaker: Kuhner Lab Shaker LSR-V-12.5, shaking diameter d0 = 25 mm - Sulfite-based: 12 mM degassed phosphate buffer (0.5 M Na2HPO4·2H2O and 0.5 M NaH2PO4·2H2O (Roth, Karlsruhe, Germany) with 0.5 M NaSO3 (Bernd Kraft, Duisburg, Germany), 2.4*10 -5 Bromothymol blue M (Sigma-Aldrich, Steinheim, Germany), pH 8 with 30% H2SO4 (w / w) (Bernd Kraft, Duisburg, Germany), 10 -7 M's CoSO4·7H2O (Sigma-Aldrich, Steinheim, Germany) - Sterilization closure: Thomson Ultra Yield Flask AirOTop Enhanced Seal for Ultra Yield 2.5L flasks (Thomson Instrument Company, Oceanside, USA) - Camera: Samsung Galaxy A3 (2016) with the "IntervalCam" application.

[0045]

number

[0046] In the figures showing the results of this experimental example (Figs. 9 to 11), the internal structure is referred to as a "tube". OTR max : Maximum oxygen transfer rate (volume)

[0047] Experimental Example 2: OTR L by filling volume V max measurement In the first method, oxygen transfer to the liquid is considered according to the filling volume of the bioreactor vessel having the above dimensions. Since oxygen transfer mainly occurs at the liquid surface in contact with the gas, the oxygen transfer volume decreases as the volume increases. (As long as the entire bottom of the container is covered), when a liquid is supplied to a container of the same size, the smaller the volume of the liquid, the larger the ratio of the liquid surface to the volume of the liquid. Examine how the container design affects the oxygen transfer volume.

[0048] (For each container design A, B, C) The following samples were tested 1. VL = 5 mL, shaken at 250 rpm 2. VL = 10 mL, shaken at 250 rpm 3. VL = 15 mL, shaken at 250 rpm 4. VL = 10 mL, shaken at 300 rpm 5. VL = 15 mL, shaken at 300 rpm

[0049] Fig. 9 shows the results of oxygen transfer volume according to the container filling volume at two rotational speeds (Fig. 9A: 250 rpm, Fig. 9B: 300 rpm). As can be seen from the figure, the oxygen transfer rate increases due to the presence of the internal structure ("tube"), and when the gap between the outer wall of the container and the internal structure is narrow, the OTR max is significantly improved compared to the case where the gap is wide. When the rotational speed is increased, the oxygen transfer rate significantly increases (compare Fig. 9A and 9B).

[0050] Figure 10 shows the degree of improvement in oxygen transport obtained by incorporating an internal structure (tubing). In Figure 10A, it is clear that at 250 rpm, oxygen transport improves dramatically with increasing volume, in both containers with wide and narrow gaps. At 300 rpm, the improvement obtained is almost independent of the fill volume (see Figure 10B). Both figures show that the improvement obtained when the distance between the outer wall and the internal structure is short (narrow gap) is significantly higher than the improvement obtained when the distance is long (wide gap). However, both designs have a clear positive effect on oxygen transport compared to bioreactors without an internal structure.

[0051] Experimental Example 3: OTR with rotational speed n max Measurement Furthermore, the transfer of oxygen to the liquid was considered in relation to the rotational speed (n) of the bioreactor vessel having the above dimensions. It is generally known that as the liquid splashes and flows along the inner wall of the vessel, a film is formed on the inner surface of the wall, increasing the liquid surface area, and therefore, as the rotational speed increases, the oxygen transfer capacity increases. Since oxygen transfer mainly occurs at the liquid surface in contact with the gas, as the liquid surface increases, gas exchange increases.

[0052] Figure 11A shows the improvement in oxygen transport for samples 2 and 4, and Figure 11B shows the improvement in oxygen transport for samples 3 and 5. As can be seen from the figures, providing an internal structure that results in an increased surface area increases oxygen transport into the liquid at both rotational speeds. The stronger effect of narrowing the gap can be explained by increasing the area of ​​the added inner surface (inner surface of the internal structure) for forming a liquid film. The dimensions and design of the bioreactor vessels shown in the experimental examples above should be considered illustrative. Specific sizes as defined herein are modifiable. In fact, bioreactor vessels of larger or smaller dimensions can be fabricated, but it is particularly preferable that the dimensional ratios (in particular, the ratios of walls, spaces, cross-sections, and openings) essentially correspond to the dimensions described herein.

Claims

1. A bioreactor container (1) having an outer wall (2) and a bottom (3), further comprising at least one integrated internal structure (4) that provides at least two additional surfaces (4a), (4b) to the internal space of the container, The internal structure (4) is spaced apart from the outer wall (2) of the container and is in contact with the bottom (3). The internal structure (4) is a bioreactor container (1) for biological cell cultures in which the cell culture is shaken, having a wall comprising an outer surface (4a) and an inner surface (4b), and having at least one opening (5) at its lower end.

2. The bioreactor container according to claim 1, wherein the opening (5) has a dimension of at least 0.05 mm in any direction, depending on the total dimensions of the bioreactor container.

3. The bioreactor container according to claim 1, wherein the opening (5) is any shape selected from a circle, a semicircle representing a sector, an ellipse, a semiellipse representing an egg shape, a triangle, a rectangle, a polygon, a wavy shape, or a combination thereof.

4. The bioreactor container according to claim 1, wherein the outer wall (2) and the internal structure (4) of the container have basically the same shape, and the shape is conical, cylindrical, or elliptical.

5. The bioreactor container according to claim 1, having a plurality of the aforementioned internal structures (4), thereby providing more than two additional surfaces within it.

6. The bioreactor container according to claim 1, wherein the outer surface (4a) of the internal structure (4) is spaced at least 1 / 15 of the cross-section of the entire reactor container from the inner surface (2b) of the container wall (2).

7. The outer surface (4a) of the internal structure (4) is the cross section (CS) of the outer wall of the container. оｕｔ Cross-section of the internal structure relative to (CS ｉｎ The outer wall (2) of the container is spaced apart from the inner surface (2b) by a distance such that the ratio of ) is in the range of 0.95 to 0.

4. The bioreactor container according to claim 1, wherein the cross-section is measured along the bottom (3) in each of the inner surface (2b) and inner surface (4b) spaces.

8. The bioreactor container according to claim 1, further comprising a cover (6).

9. The bioreactor container according to claim 1, further having an inlet and / or outlet, thereby enabling the addition or recovery of the contents of the container, or the placement of a measuring device inside the container.

10. Use of the bioreactor container according to claim 1 for growing biological cells, or animal cells, human cells, or plant cells.

11. A method for growing biological cells under specified gas conditions, comprising placing cells to be grown into a bioreactor container (1) according to claim 1, filling the container at least partially with liquid, and shaking the container so that at least the liquid rotates within the container.

Images