Bioreactors capable of operating in static and dynamic modes and methods of use thereof

Abstract

The present application describes a bioreactor comprising a gas permeable membrane disposed on a housing, the gas permeable membrane at least partially covering a transfer opening. The bioreactor is also provided with a mixing element and a port adapted to receive a sensor that measures a parameter value related to a process carried out in the bioreactor. The bioreactor includes at least one opening in one of the walls. The at least one opening is covered by a gas permeable membrane.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 406,967, filed on September 15, 2022, which is incorporated herein by specific reference. Background Art 1. Technical Field

[0003] The present invention relates to bioreactors capable of operating in a variety of cell culture modes and bioreactor orientations to maximize and optimize cell expansion, cell preservation, and cell processing within a single bioreactor vessel. For example, the bioreactors of the present disclosure can be operated in a static mode in which no mechanical mixing occurs and a dynamic mode in which any degree of mechanical mixing occurs and / or alternating operating modes and within each cell culture mode in a vertical orientation, a horizontal orientation, or an angled orientation.

[0004] 2. Related technologies

[0005] The expansion of biological cells in bioreactors requires key control of many different process parameters. For example, as cells expand, they absorb oxygen and release CO2 from the surrounding medium. The concentration of oxygen and CO2 in the medium must be carefully monitored and regulated to ensure the viability and optimal expansion of the cells. Other processing parameters that are usually monitored and controlled include pH value, temperature and cell density. As the cell colony increases, in order to help maintain the correct control of various processing parameters, cells are usually cultivated by sequentially transferring cells to larger and larger bioreactors. For example, cell seed cultures can initially be started by culturing in small flasks, which are moved on a vibration table to keep the cell suspension uniformly mixed. Once the cell density reaches a predetermined value, the cell suspension is transferred to a larger benchtop bioreactor, in which the suspension is mixed with an additional medium. In order to maintain the correct mixing and oxygenation of a larger volume of cell suspension in a benchtop bioreactor, a benchtop bioreactor is usually equipped with an internal impeller for mechanical mixing and a sparger for delivering gas to the compartment. Then, once the cell density increases to a predetermined value again, the cell suspension can be transferred to a larger production bioreactor for further expansion with additional media.

[0006] Although the time-tested method of sequentially transferring and expanding cell populations to increasingly larger bioreactors to achieve the desired cell production is effective, this method has many disadvantages. For example, transferring cells between different bioreactors is time-consuming and labor-intensive. In addition, the cell suspension must always be kept sterile, which generally requires strict validation requirements. Transferring cell suspensions between different bioreactors increases the risk of contamination. In addition, using multiple different bioreactors to expand single cell batches is expensive in terms of the cost, operation, storage, and maintenance of multiple different types, sizes, and / or designs of bioreactors. There are also other disadvantages.

[0007] Therefore, what is needed in the art are new bioreactors and related operating methods that minimize some or all of the above-mentioned disadvantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Various embodiments of the invention will now be discussed with reference to the accompanying drawings. It should be appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope.

[0009] Figure 1 A front perspective view of the bioreactor.

[0010] Figure 2 for Figure 1 A rear perspective view of the bioreactor is shown.

[0011] Figure 3 for Figure 1 A partially exploded view of the bioreactor is shown.

[0012] Figure 4 Its surrounding side walls are removed Figure 1 A perspective view of the bioreactor is shown.

[0013] Figure 5 for Figure 1 A subassembly of a bioreactor is shown, illustrating the drive shaft and mixing elements thereon.

[0014] Figure 6 for Figure 5 An enlarged cross-sectional view of the terminal end of the drive shaft shown engaging a secure support.

[0015] Figure 7 For multiple units stacked on a shelf (such as in an incubator) Figure 1 A perspective view of the bioreactor is shown.

[0016] Figure 8 for Figure 5 A perspective view of an alternative embodiment of a drive shaft is shown having a free floating terminal end and an alternative sparger that may be used in a bioreactor.

[0017] Fig. 9 for Figure 1 A partially exploded perspective view of an alternative embodiment of a bioreactor is shown in which a second transfer opening is formed in the bottom end wall and is covered by a second gas permeable membrane.

[0018] Fig.10 for Figure 5 A perspective view of an alternative embodiment of a drive shaft is shown, the drive shaft comprising adjacent drive shaft sections, each drive shaft section having a helical configuration.

[0019] Fig.11 is a left side perspective view of an alternative embodiment of a bioreactor system.

[0020] Fig.12 for Fig.11 A right side perspective view of the bioreactor system is shown.

[0021] Fig.13 for Fig.11 An enlarged perspective view of the bottom end wall of the bioreactor is shown.

[0022] Fig.14 for Fig.11 An enlarged perspective view of the top wall of the bioreactor is shown.

[0023] Fig.15 for Fig.14 An enlarged bottom perspective view of the top end wall is shown.

[0024] Fig.16 for Fig.11 A top perspective view of the heater support is shown.

[0025] Fig.17 for Fig.16 Bottom perspective view of the heater support shown.

[0026] Fig.18 for Fig.11 An exploded view of an alternative embodiment of a support housing is shown.

[0027] Fig.19 is a perspective view of another alternative embodiment of a bioreactor system.

[0028] Fig. 20 is a perspective view of a heating jacket that may be used with the bioreactors disclosed herein.

[0029] Fig.21 is a perspective view of a bioreactor according to an example embodiment.

[0030] Fig. 22 for Fig.21 Top view of the bottom of the bioreactor's top cap.

[0031] Fig.23A An impeller assembly according to an example embodiment is depicted.

[0032] Fig. 23B is a cross-sectional view of an impeller mounting hub according to an example embodiment.

[0033] FIG. 24A to FIG. 24B Depicted are top and perspective views of a bioreactor base according to example embodiments.

[0034] FIG. 25A to FIG. 25B Depicted are a perspective view of a bioreactor and a top view of the base of the bioreactor according to an example embodiment.

[0035] Fig.26 A sprinkler according to an example embodiment is depicted.

[0036] Fig. 27 A bioreactor base is depicted according to an example embodiment.

[0037] FIG. 28A to FIG. 28C Sensors and port groups are depicted according to example embodiments.

[0038] FIG. 29A to FIG. 29B Depicted are front and rear views of a reactor pod according to an example embodiment.

[0039] Fig.30 Detailed description of the invention A bioreactor chamber wall according to an example embodiment.

[0040] FIG. 31A to FIG. 31D Depicted are front, back, and cross-sectional views of a sensor group according to an example embodiment.

[0041] Fig.32 is the top wall of a bioreactor according to an example embodiment.

[0042] FIG. 33A to FIG. 33E Depicted is a series of impellers according to example embodiments.

[0043] FIG. 34A to FIG. 34C Depicted are perspective, cross-sectional, and partial top views of an impeller mounting hub according to an example embodiment.

[0044] FIG. 35A to FIG. 35B Depicted are perspective and cross-sectional views of an impeller assembly according to an example embodiment.

[0045] Fig.36 An autologous cell therapy system and process flow including one or more dual-mode bioreactors or bioreactor cassettes according to example embodiments is depicted. DETAILED DESCRIPTION

[0046] Before describing the present disclosure in detail, it should be understood that the present disclosure is not limited to the specific exemplified equipment, systems, methods or process parameters, which of course can be changed. It should also be understood that the terms used herein are only used for the purpose of describing the specific exemplary embodiments of the present disclosure, and are not intended to limit the scope of the present disclosure in any way.

[0047] The term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0048] It should be noted that throughout this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "port" includes one, two, or more ports.

[0049] As used in the specification and the appended claims, directional terms herein, such as "top", "bottom", "left", "right", "up", "down", "upper", "lower", "proximal", "distal", etc., are used only to indicate relative directions and are not intended to otherwise limit the scope of the present disclosure or the claims.

[0050] Where possible, similar reference numerals are used in the various drawings. In addition, multiple instances of sub-elements of elements and / or parent elements may each contain separate letters attached to the element number. For example, two instances of a specific element "10" may be labeled as "10A" and "10B". In the case described, element markings (e.g., "10") without additional letters may be used to generally refer to an instance of an element or any one of these elements. Element markings (e.g., "10A") including additional letters may be used to refer to a specific instance of an element or to distinguish or emphasize the multiple uses of an element. In addition, element markings with additional letters may be used to refer to alternative designs, structures, functions, specific implementations and / or embodiments of an element. For example, two alternative exemplary embodiments of a specific element may be labeled as "10A" and "10B". In this case, element markings (e.g., "10") without additional letters may be used to generally refer to all instances of alternative embodiments or any one of these alternative embodiments.

[0051] Various aspects of the apparatus and systems of the present invention may be presented by describing multiple components that are coupled, attached, and / or coupled together. As used herein, the terms "coupled," "attached," and / or "coupled" are used to indicate a direct connection between two components, or, where appropriate, an indirect connection to one another through an intervening component or intermediate component. In contrast, when a component is referred to as being "directly coupled," "directly attached," and / or "directly coupled" to another component, there are no intervening elements. Furthermore, as used herein, the terms "connected," "connected," and the like do not necessarily imply direct contact between two or more elements.

[0052] As used herein, the term "gas permeable membrane" is a layer (e.g., a solid layer or a non-fluid layer) that allows gas to pass through. More specifically, a "gas permeable membrane" can be a membrane that allows various gas molecules (including oxygen, carbon dioxide, and nitrogen) to pass through the membrane due to pressure, partial pressure, or concentration differences across the membrane. However, the gas permeability of the membrane does not allow airflow or visible bubbles to pass through. In an exemplary embodiment, the gas permeability of the membrane at 23 degrees Celsius and 1 bar can be 500 mL / (m 2 *day) and 25,000mL / (m 2 *day) in the range of 5,000mL / (m 2 *day) and 10,000mL / (m 2 * day) is more preferred. The gas permeability is also generally less than 75,000 mL / (m 2 *day)、100,000mL / (m 2 *day), 125,000mL / (m 2 *day) or 150,000mL / (m 2 * days). Gases permeable to these membranes include, for example, O2, CO2, and N2. Gas permeable silicone (e.g., dimethyl silicone) membranes of approximately 0.005 to 0.007 inches thick may be used, and are mentioned in U.S. Pat. No. 9,567,565, which is hereby incorporated by specific reference. Example gas permeable membranes include Series, which are available from Wilson Wolf Corporation, 335th Ave NW, Saint Paul, MN 55112 (see, e.g., P / N 85500S-CS and 81100S). Other examples of gas permeable membranes and devices containing gas permeable membranes are gas permeable plates available from Coy Lab Products (see catalog number 8602000). These plates allow control of the O2 level in contact with cells in an incubator. The specifications of these plates are as follows: 25 μm polymer film, which allows for higher gas transmission rates while retaining liquids, O2 permeability greater than 9000 cm 3 / M 2 , CO2 permeability greater than 7000cm 3 / M 2 .

[0053] As used herein, the terms "expanded" and "expand" refer to cell multiplication. For example, if the number of cells in a culture is increased from 1,000 cells to 4,000 cells, the cells will have expanded fourfold. Assuming that 100% of the cells in the culture are multiplying and multiplying at the same rate, this amount of expansion will occur after two cell divisions. In many instances herein, the terms "cultured" and "expanded" are used interchangeably.

[0054] As used herein, the term "activation" refers to a cell state after sufficient cell surface moieties are attached to induce measurable morphological, phenotypic and / or functional changes. In the context of T cells, such activation may be a state of T cells that have been sufficiently stimulated to induce cell proliferation. The activation of T cells may also induce the production and / or secretion of cytokines, as well as the upregulation or downregulation of the expression of cell surface molecules (such as receptors or adhesion molecules), or the upregulation or downregulation of the secretion of certain molecules and the performance of regulatory or cytolytic effector functions. In the context of other cells, this term may infer the upregulation or downregulation of specific physicochemical processes.

[0055] In some instances, stimulation may include the primary response induced by the connection of the cell surface portion. For example, in the context of a receptor, this stimulation may require the connection of a receptor and subsequent signaling events. In some instances, the amplification of T cells may, for example, include stimulating these T cells. Regarding the stimulation of T cells, this stimulation may refer to the connection of the T cell surface portion of the subsequent induction of signaling events (such as in conjunction with TCR / CD3 complexes) in the embodiments. In some instances, the stimulation event may activate cells and raise or lower the expression of cell surface molecules (such as receptors or adhesion molecules), or raise or lower the secretion of molecules, such as lowering tumor growth factor β (TGF-β) or raising IL-2, IFN-γ, etc. In some instances, even if there is no direct signaling event, the connection of the cell surface portion may also lead to the reorganization of the cytoskeleton structure or the aggregation of the cell surface portion, and each cell surface portion may be used to enhance, modify or change the subsequent cell response.

[0056] As used herein, the term "stimulatory agent" refers to a molecule that binds to one or more cell types and induces a cellular response. The agent may bind to any cell surface moiety present on the target cell population, such as a receptor, antigenic determinant, or other binding site. The agent may be a protein, a peptide, an antibody and its antibody fragments, a fusion protein, a synthetic molecule, an organic molecule (e.g., a small molecule), etc. In embodiments, antibodies are used as a prototypical example of such agents in the context of T cell stimulation.

[0057] The antibodies used in the methods described herein can be of any species, class or subtype, as long as such antibodies can react appropriately with the target of interest (e.g., CD3, TCR or CD28). Therefore, "antibodies" used in the methods described herein include:

[0058] (a) any of the various classes or subclasses of immunoglobulin (e.g., IgG, IgA, IgM, IgD or IgE derived from any animal (e.g., any of the conventionally used animals, such as sheep, rabbit, goat, mouse, camel or egg yolk)),

[0059] (b) monoclonal or polyclonal antibodies,

[0060] (c) intact antibodies or monoclonal or polyclonal antibody fragments, which fragments are those containing the antibody binding region, e.g., without the Fc portion (e.g., Fab, Fab′, F(ab′)2, scFv, V H Fv is a fragment of a heavy chain (H or other single domain antibodies), a so-called "half-molecule" fragment obtained by reductive cleavage of the disulfide bonds linking the heavy chain components of an intact antibody. Fv can be defined as a fragment containing the variable regions of a light chain and a heavy chain and expressed as two chains.

[0061] (d) Antibodies produced or modified by recombinant DNA or other synthetic techniques, including monoclonal antibodies, antibody fragments, "humanized antibodies", chimeric antibodies, or synthetically prepared or altered antibody-like structures.

[0062] Also included are functional derivatives or "equivalents" of antibodies, for example, single chain antibodies, CDR-grafted antibodies, etc. Single chain antibodies (SCAs) can be defined as genetically engineered molecules containing the variable region of a light chain and the variable region of a heavy chain connected by a suitable polypeptide linker into a fused single chain molecule.

[0063] As used herein, "chimeric antigen receptor" or "CAR" or "CARs" refers to an engineered receptor that is transplanted antigen specificity into a cell (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof). CAR is also referred to as an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor. In an embodiment, CAR includes one or more antigen-specific targeting domains, an extracellular domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain. In an embodiment, if CAR targets two different antigens, the antigen-specific targeting domains can be arranged in series. In an embodiment, if CAR targets two different antigens, the antigen-specific targeting domains can be arranged in series and separated by a linker sequence.

[0064] CAR is an engineered receptor that transplants any specificity onto immune cells (e.g., T cells, such as activated T cells). These receptors are used to transplant the specificity of monoclonal antibodies onto immune cells; the transfer of their coding sequences is promoted by retroviral vectors. Receptors are called chimeras because they are composed of parts from different sources. CAR can be used as a treatment for cancer by adoptive cell transfer. T cells are removed from patients and modified so that they express receptors specific to the patient's specific cancer. T cells that recognize and kill cancer cells are reintroduced into the patient. In embodiments, modification of T cells derived from donors other than patients can be used to treat patients.

[0065] Using adoptive transfer of T cells expressing chimeric antigen receptors, CAR-modified T cells can be engineered to target any tumor-associated antigen. After the patient's T cells are collected, the cells are genetically engineered to express a CAR specifically for an antigen on the patient's tumor cells and then infused back into the patient.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although a variety of methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, preferred materials and methods are described herein.

[0067] Various aspects of the devices, systems, and methods of the present invention may be presented with reference to one or more exemplary embodiments. As used herein, the terms "exemplary," "embodiment," and "exemplary embodiment" mean "serving as an example, instance, or illustration," and should not necessarily be construed as required, or preferred or advantageous over other embodiments disclosed herein.

[0068] The present invention relates to a bioreactor for amplifying cells present in a suspension that is dispensed into or formed within a bioreactor. In an exemplary embodiment, the cells are human cells, such as T cells, for cell therapy. The bioreactor can operate in both a static mode and a dynamic mode, in which the mixer of the bioreactor is stationary and / or does not mix the suspension during a specific growth phase (e.g., the first phase, after electroporation, or other phases in which cells are more susceptible to damage), and in a dynamic mode, minimal, continuous, substantially continuous, low or high mixing (such as rpm (RPM) mixing) can be used to mix the suspension during the second growth phase.

[0069] The bioreactors described herein can be operated by rotating the mixing element at various RPMs. The number of RPMs used will generally vary with the type / configuration of the mixing element and the type and condition of the cells in the bioreactor. This is in part because some cells are more tolerant of mechanical shearing than other cells. In general, the mixing element RPM (such as an impeller) will typically vary from about 40 RPM to about 1,500 RPM (e.g., about 40 RPM to about 1,000 RPM, about 40 RPM to about 800 RPM, about 40 RPM to about 600 RPM, about 40 RPM to about 400 RPM, about 40 RPM to about 300 RPM, about 40 RPM to about 200 RPM, about 100 RPM to about 1,000 RPM, about 100 RPM to about 800 RPM, about 100 RPM to about 600 RPM, about 100 RPM to about 400 RPM, about 100 RPM to about 300 RPM, about 60 RPM to about 1,000 RPM, about 60 RPM to about 300 RPM, etc.). For more sensitive process conditions or when cell types or conditions are fragile (eg, after electroporation), the bioreactor and mixer may be operated in dynamic mode at lower RPMs of less than 40 RPM or less than 10 RPM.

[0070] Figure 1 and Figure 2One embodiment of a bioreactor 10 incorporating features of the present disclosure is depicted in . The bioreactor 10 includes a housing 12 having a top end wall 14, an opposite bottom end wall 16, and a surrounding side wall 18 extending therebetween. The surrounding side wall 18 has a front face 20, an opposite rear face 22, and opposite side faces 24 and 26, each extending between the top end wall 14 and the bottom end wall 16.

[0071] In the depicted embodiment, the surrounding sidewall 18 has a rectangular or square transverse cross-section. However, in other embodiments, the surrounding sidewall 18 may have other transverse cross-sectional configurations, such as circular, oval, or polygonal. More specifically, the surrounding sidewall 18 is depicted as including a front wall 28, a rear wall 30, and opposing side walls 32 and 34, the front wall including the front face 20, the rear wall including the rear face 22, and the opposing side walls extending between the front wall 28 and the rear wall 30. The side walls 32 and 34 include the sides 24 and 26, respectively.

[0072] Go to Figure 3 , shell 12 has the inner surface 36 that limits compartment 38.For the convenience of correct operation, compartment 38 is specially designed to be relatively small.For example, the volume of compartment 38 is generally at least or less than 50 milliliters, 100 milliliters, 250 milliliters, 500 milliliters, 1 liter, 5 liters, 10 liters, 20 liters, 30 liters, 40 liters, 50 liters or in the scope between any two of the aforementioned.For example, compartment 38 has the volume in the scope between 250 milliliters and 50 liters usually, wherein between 1 liter and 20 liters or between 1 liter and 10 liters is more common.Also can use other volumes.In one embodiment, shell 12 and wall thereof are made of gas and liquid (such as medium) impermeable material.

[0073] In addition, shell 12 and its wall are generally rigid.For example, in an exemplary embodiment, shell 12 is sufficiently rigid so that when compartment 38 is full of liquid (such as water or medium), shell will not bend, flex and / or expand.Shell 12 is usually made of plastics such as polycarbonate, polyolefin, polyester, polystyrene and polyacrylic acid, and can be produced by a molding process such as injection molding, extrusion, blow molding, 3d printing (additive manufacturing), rotational molding or any combination thereof.Forming shell 12 by plastic also makes shell relatively cheap so that it can be discarded or recycled after single use.The rigidity of shell 12 provides stability for bioreactor 10, and enables it to be self-supported during its different operating modes to carry out correct operation, as discussed below.However, in alternative embodiments, shell 12 can be formed by a material that has a certain degree of bending, flexing and / or expansion during use but still has sufficient rigidity to self-support. In other alternative embodiments, as further discussed below, the housing 12 may include a collapsible bag made from one or more polymer film sheets supported within a self-supporting, reusable support housing, such as may be made from the same material and have the same properties as the housing 12 discussed above.

[0074] Also like Figure 3 As shown, a first transfer opening 40 is formed on the front wall 28 / front face 20 and extends through the front wall / front face so as to communicate with the compartment 38. As discussed below, the first transfer opening 40 is used to enable gas to be transferred into and out of the compartment 38, and more specifically, to be transferred into and out of the suspension contained therein, particularly when the bioreactor 10 is used in a static mode. The first transfer opening 40 needs to be large enough to facilitate the transfer of the required gases (particularly CO2 and oxygen) to the cells within the compartment 38 to keep the cells healthy and expanding. In one embodiment, the front wall 28 / front face 20 has an outer surface 42 with an area that includes the area through which the first transfer opening 40 passes. In an exemplary embodiment, the area of ​​the first transfer opening 40 is generally at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the area of ​​the outer surface 42, or in a range between any two of the foregoing percentages.

[0075] Back to Figure 1, shell 12 is elongated, and its height is greater than width. Specifically, shell 12 has width W or diameter and the height H extending between top end wall 14 and bottom end wall 16. Width W can extend between sidewall 32 and 34, i.e. the width of front wall 28, or extend between front wall 28 and rear wall 30. Height and width W can vary significantly according to the selected volume for compartment 38. In some common embodiments, height H is at least or less than 0.2 meter, 0.3 meter, 0.4 meter, 0.6 meter, 0.8 meter, 1 meter or the scope between any two of the foregoing items. In some exemplary embodiments, maximum or minimum width W or diameter is generally at least or greater than 2.5cm, 5cm, 7.5cm, 10cm, 15cm, 20cm, 30cm, 40cm, 50cm or the scope between any two of the foregoing items. In one embodiment, the maximum height H is at least 1.2 times, 1.4 times, 1.6 times, 1.8 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, or in the range between any two of the aforementioned values, the maximum width W or diameter. Similarly, other sizes may also be used depending on the intended application. As discussed further below, this elongated configuration may also help optimize mixing efficiency and spraying efficiency when in dynamic mode.

[0076] refer to Figure 1 and Figure 3, the bioreactor 10 also includes a gas permeable membrane 50, which is disposed on the housing 12 so as to cover at least a portion of the first transfer opening 40. The gas permeable membrane 50 allows gas (particularly oxygen and CO2) to be transferred through it. Although not required, the gas permeable membrane 50 is generally liquid-impermeable so that liquid cannot leak through, and more specifically, is impermeable to the medium used in the cell suspension. That is, although water vapor can penetrate through the gas permeable membrane 50, liquids generally cannot. For example, in an exemplary embodiment, when the compartment 38 is filled with liquid (such as a medium or a cell suspension), under ambient conditions, i.e., when no external force is applied to the liquid, the liquid will not leak through the gas permeable membrane 50. Thus, the gas permeable membrane 50 helps keep the compartment 38 sterile by preventing contaminants from passing through it. Generally speaking, the gas permeable membrane 50 needs to be sufficiently permeable so that, based on the oxygen concentration gradient between the oxygen within the suspension contained in the housing 12 / compartment 38 and the oxygen in the surrounding environment, oxygen can pass through the gas permeable membrane 50 and into the suspension to oxygenate the cells. The same is true for CO2, which must be able to pass through the gas permeable membrane 50. Therefore, in some embodiments, the gas permeable membrane 50 is a diffusion gas permeable membrane that only allows gas to pass through it by passive gas exchange through diffusion. That is, in some embodiments, there are no pores in the gas permeable membrane 50, and the gas can only pass through the gas permeable membrane 50 at the molecular level by applying a diffusion gradient across the gas permeable membrane 50.

[0077] In an exemplary embodiment, the gas permeability of the gas permeable membrane 50 may be 500 mL / (m 2 *day) and 25,000mL / (m 2 *day) in the range of 5,000mL / (m 2 *day) and 10,000mL / (m 2 * day) is more preferred. The gas permeability is also generally less than 75,000 mL / (m 2 *day)、100,000mL / (m 2 *day), 125,000mL / (m 2 *day) or 150,000mL / (m 2 * days). In an exemplary embodiment, the gas permeable membrane 50 comprises a sheet or film of gas permeable silicone, dimethyl silicone, expanded polytetrafluoroethylene (ePTFE), FEP, or a fluoropolymer. Examples of other materials and properties of the gas permeable membrane 50 are those discussed previously with respect to the definition of "gas permeable membrane". One specific example of a material that can be purchased and used as the gas permeable membrane 50 is 80M Other materials known in the art may also be used.In some embodiments, the membrane is a single sheet of material.In some embodiments, the gas permeable membrane may contain internal support structures and materials that do not necessarily contribute to gas permeability but do provide physical strength or support structure.

[0078] The gas permeable membrane 50 is attached to the housing 12 so as to seal and close the first transfer opening 40 so that liquid cannot pass through it. The gas permeable membrane 50 can be attached to the housing 12 in a variety of different ways. For example, the gas permeable membrane can be directly fixed to the housing 12 by welding or adhesive. However, due to the incompatibility between the materials, it may be difficult to directly connect the gas permeable membrane 50 to the housing 12. Therefore, in an exemplary embodiment, a support frame can be used to connect the gas permeable membrane 50 to the housing 12.

[0079] Specifically, the gas permeable membrane 50 is shown as having an outer side 52 and an opposite inner side 54 that each extend to surround a peripheral edge 56. The support frame 58 has a front face 60, an opposite rear face 62, and an inner surface 64 that surrounds a passage 66 extending between the front face 60 and the rear face 62. The inner surface 64 has an annular recess 68 formed thereon that is configured to receive the peripheral edge 56 of the gas permeable membrane 50 so as to form a liquid-tight seal therebetween. For example, the peripheral edge 56 of the gas permeable membrane 50 may be sealed within the recess 68 by adhesive, press fit, welding, crimping, or other conventional techniques, depending on the material properties of the support frame 58 and the gas permeable membrane 50. In other embodiments, the support frame 58 may include two overlapping layers, wherein the peripheral edge 56 of the gas permeable membrane 50 is sandwiched and fixed between the two overlapping layers. The support frame 58 is generally made of a material suitable for attachment to the housing 12. For example, in an exemplary embodiment, the rear face 62 of the support frame 58 can be fixed to the exterior surface 42 of the front wall 28, such as by using welding, adhesives, fasteners, etc. The advantage of welding is that it eliminates possible contamination caused by adhesives and generally seals better than fasteners. In other embodiments, the support frame 58 can be fixed directly within the first transfer opening 40.

[0080] In one embodiment, the support frame 58 is made of the same material as discussed above with respect to the housing 12. In the case where the housing 12 includes a collapsible bag (as previously cited), the gas permeable membrane 50 can be secured to the bag so as to cover the opening through the bag wall. The gas permeable membrane 50 can be secured to the bag by first securing the gas permeable membrane 50 to the support frame (as previously discussed) and then securing the support frame to the collapsible bag; or by directly welding or otherwise securing the gas permeable membrane 50 to the bag. In some embodiments, the support frame can include a gap support structure attached beyond the peripheral edge to facilitate support and positioning of the membrane. The support frame can also include a window with internal supports that facilitate support and positioning of the membrane.

[0081] It should be noted that the gas permeable membrane 50 is not used as a sprinkler. That is, in an exemplary embodiment, the permeability of the gas permeable membrane does not allow bubbles to pass through it. In addition, the gas permeable membrane 50 is generally in the form of a flat plate or sheet, wherein the inner side 54 faces the compartment 38 and is directly connected to the compartment, and the outer side 52 faces the environment outside the compartment 38 and is directly connected to the environment outside the compartment. In addition, the gas permeable membrane 50 is generally free of any internal compartments that can deliver the spraying gas, and is generally not directly connected to any tubular member that can deliver the spraying gas to the gas permeable membrane 50. In addition, the gas permeable membrane 50 is generally in the form of a single continuous plate or sheet, rather than two or more separate panels or sheets that are overlapped and connected together.

[0082] The present disclosure also includes means for stacking a plurality of bioreactors 10 on top of each other when the bioreactors 10 are arranged horizontally or vertically in a static mode or a dynamic mode so that gaps are formed between the bioreactors. Figure 1 and Figure 2In one embodiment shown, a plurality of first mounting portions 80 are formed on the front wall 28, and a plurality of spaced apart second mounting portions 82 are formed on the rear wall 30. The mounting portions 80 and 82 are configured so that for a plurality of identical bioreactors 10, the first mounting portion 80 of the first bioreactor 10 can be engaged with the second mounting portion 82 of the second bioreactor 10 to form a secure engagement between the bioreactors and provide spacing between adjacent bioreactors. It should be understood that the mounting portions 80 and 82 can have a variety of different configurations. In the depicted embodiment, the first mounting portion 80 includes four independent and spaced apart first mounting portions 80A-80D protruding outward from the front wall 28. In one embodiment, the first mounting portions 80A-80D are disposed at or near the four corners of the front wall 28. Each first mounting portion 80A-80D includes a body 84 having a recess 86 formed at its terminal face. The second mounting portion 82 also includes four mounting portions 82A-82D protruding outward from the rear wall 30. The second mounting features 82A- 82D may be disposed at or near a corner of the rear wall 30 , but in any event are positioned so that they align with the first mounting features 80A- 80D of an adjacent bioreactor 10 .

[0083] Each second mounting portion 82A-82D terminates at an end 88 that is configured to be received within a recess 86 of a first mounting portion 80A-80D. Figure 7 , during use, as will be discussed in more detail below, multiple identical bioreactors 10A, 10B, and 10C can be stacked on top of each other when horizontally oriented by coupling a first mounting portion 80A-80D with a second mounting portion 82A-82D of an adjacent bioreactor. In alternative embodiments, the first mounting portions 80A-80D and the second mounting portions 82A-82D can be releasably locked together, such as by using one or more fasteners or latches, to further increase stability and prevent undesired separation.

[0084] The connection between the mounting portions 80 and 82 provides a secure and stable assembly of multiple bioreactors, and also provides spacing between bioreactors so that gas can flow freely through the gas permeable membrane 50. The mounting portion 80 also provides a support for the lowermost bioreactor 10 to support the housing 12 of the lowermost bioreactor 10 away from the surface on which the mounting portion 80 rests. Likewise, as discussed below, the spacing enables gas to flow freely through the gas permeable membrane 50. It should be understood that the mounting portions 80 and 82 can have a variety of different configurations. For example, the mounting portions 80 and 82 can be reversed. In other embodiments, the mounting portion protrudes outward from one of the front wall 28 or the rear wall 30, and the mounting portion on the other of the front wall 28 or the rear wall 30 may include a recess configured to receive the other mounting portion. In other embodiments, only one of the front wall 28 or the rear wall 30 may require a mounting portion, wherein these mounting portions are configured to be securely engaged with the housing 12 of the adjacent barrier without engaging with other mounting portions. In other embodiments, the mounting portion does not need to be formed directly on the housing 12. For example, a separate and removable bracket / mounting portion may be placed between adjacent bioreactors 10 to facilitate secure stacking and spacing therebetween. In other embodiments, mounting portions 80 and 82, or alternatives thereto as discussed above, may also be positioned at the top end wall 14 and / or the bottom end wall 16 to enable vertical stacking of bioreactors 10 when the bioreactors 10 are in a vertical orientation. This may be particularly useful when a gas permeable membrane is placed on the bottom end wall 16 of the bioreactor 10, as discussed below with respect to Fig. 9 Other configurations may also be used.

[0085] In the depicted embodiment, the top end wall 14 is formed separately from the surrounding side wall 18 and is configured to be mounted at its upper end. Figure 4 , the top end wall 14 generally includes an exterior surface 264 and an opposing interior surface 265. The top end wall 14 is mounted at the upper end of the surrounding side wall 18 so that the interior surface 265 faces the compartment 38. More specifically, in an exemplary embodiment, the top end wall 14 includes a top panel 266 having an exterior surface 264 and an opposing interior surface 265, each of which extends to a peripheral edge 272. A mounting lip 274 protrudes downwardly from the interior surface 265 so as to be slightly inset from the peripheral edge 272 and form a continuous ring. Reference Figure 1 and Figure 4 During assembly, the mounting lip 274 slides into the opening 276 formed at the upper end 278 of the surrounding side wall 18, while the inner surface 265 of the top panel 266 is located at the upper edge 277 of the surrounding side wall 18 (see Fig.18). The mounting lip 274 is sealed to the interior surface of the surrounding side wall 18 to close the opening 276. The sealing joint can be achieved by welding, adhesives, press-fit connections, use of sealing rings or other conventional techniques. The above-described configuration of the top end wall 14 simplifies the production of both the top end wall 14 and the surrounding side wall 18, and provides easy engagement therebetween. However, it should be understood that the top end wall 14 may also have a variety of other configurations for mounting at the upper end 278 of the surrounding side wall 18.

[0086] Ports 105 and 106 are also shown formed on top end wall 14 and in communication with chamber 38. Tubes 108 and 110 are coupled to ports 105 and 106, respectively, and project from top end wall 14 toward bottom end wall 16. Tubes 108 and 110 may be used in conjunction with ports 105 and 106 to deliver fluids and / or components into compartment 38 and / or remove all or some suspension from compartment 38. During operation, ports 105 and 106 are either closed or coupled to additional conduits.

[0087] Go to Figure 4 and Figure 5 , the bioreactor 10 also includes a first sensor 94 and a spaced second sensor 96. The sensors 94 and 96 are mounted on the top end wall 14 and protrude downwardly into the compartment 38 toward the bottom end wall 16. The sensors 94 and 96 may each include sensors common to other conventional bioreactors, such as pressure sensors, temperature sensors, foam sensors, glucose sensors, pH sensors, DO sensors, CO2 sensors, density sensors, cell density sensors, etc. Although two sensors 94 and 96 are shown, in alternative embodiments, the bioreactor 10 may have at least 2, 3, 4 or more sensors mounted on the housing 12 (such as the top end wall 14) and protruding in the compartment 38. For example, the bioreactor 10 typically includes a temperature sensor, a pH sensor, a DO sensor, and a CO2 sensor. Other sensors may also be added. As will be discussed in more detail below, it should be understood that the sensors may have a variety of different configurations and may be placed in a variety of different positions on the housing 12.

[0088] The bioreactor 10 also includes a gas filter 100 that is in communication with the compartment 38. Specifically, in an exemplary embodiment, a port 102 is formed on the top wall 14 that is in communication with the compartment 38. A tube 104 is connected to the port 102 and extends to the gas filter 100. As will be discussed in more detail below, during operation in a dynamic mode, gas is sprayed into the compartment 38 and passes through the suspension. The gas filter 100 acts as a one-way valve that enables gas to leave the compartment 38 while also preventing contaminants from entering the compartment 38. Specifically, in one embodiment, the gas filter 100 is a sterilizing filter with a pore size that is small enough to prevent contaminants from passing through it but allows gas to escape through it. In the depicted embodiment, a hanger 112 is optionally mounted to the housing 12 / top wall 14 and is used to support the tube 104 / gas filter 100, thereby stabilizing the gas filter 100 and preventing the tube 104 from kinking. The hanger 112 includes a main rod 113 vertically upright from the top end wall 14 and a U-shaped support rod 114 protruding radially outward from the free end of the main rod 113. The tube 104 is received in a narrow slot defined by the support rod 114. The hanger 112 is removably received in a tubular bracket 115 protruding upward from the top end wall 14. Other forms of hangers may also be used, or alternatively, the hanger 112 may be eliminated.

[0089] like Figure 1 As shown, a plurality of optional ports 92 in communication with the compartment 38 may also be formed on the surrounding sidewall 18. For example, the ports 92 may be formed on the sidewalls 32 and / or 34 adjacent to or toward the bottom end wall 16. The ports 92 may be used to connect with tubes for flowing fluids or other components into and / or out of the compartment 38, and / or may be used to receive different types of sensors, such as those discussed above with respect to sensors 94 and 96. Depending on the orientation and mode of operation of the bioreactor 10, it may be preferred to have the ports 92 at other locations other than the top end wall 14 so that the associated tubes and / or sensors can be more effectively communicated with the suspension in the compartment 38 depending on the orientation of the bioreactor 10. The ports 92 may also be provided on any other wall surrounding the sidewall 18.

[0090] Continue to refer Figure 5 The bioreactor 10 further includes a mixing element that is removably disposed within the compartment 38 for mechanically mixing the suspension within the compartment 38 when the bioreactor is in the dynamic mode. Figure 5As shown, the drive shaft 116 is arranged to extend between a first end 118 and an opposite second end 120. The drive shaft 116 protrudes from the top end wall 14 into the compartment 38 and toward the bottom end wall 16. More specifically, in an exemplary embodiment, the first end 118 of the drive shaft 116 passes through the top end wall 14 and is rotatably coupled to the top end wall by a dynamic seal 122. The dynamic seal 122 enables the drive shaft 116 to rotate therein while preventing external contaminants from entering the compartment 38. The terminal end 124 of the first end 118 protrudes outside the dynamic seal 122 / top end wall 14. The first end 118 of the drive shaft 116 (more specifically, the terminal end 124) is coupled to a drive motor 126, which is typically electric. The drive motor 126 can be removably mounted on the housing 12, and more specifically, mounted to the top end wall 14, such as by an optional tubular protective sleeve 128 surrounding the first end 118 of the drive shaft 116. During use, a drive shaft 127 of a drive motor 126 can be removably coupled to a terminal end 124 of the drive shaft 116 such that activation of the drive motor 126 promotes rotation of the drive shaft 116. When the drive shaft 116 is not in use, the drive motor 126 and / or the protective sleeve 128 can be removed from the housing 12 to minimize the size of the bioreactor 10. For ease of production, in an exemplary embodiment, the drive shaft 116 can include an upper section 129 that passes through the dynamic seal 122 and a lower section 130 that is disposed within the compartment 38. The upper section 129 and the lower section 130 can be removably or permanently coupled together by a coupler 131 located within the compartment 38.

[0091] In an alternative exemplary embodiment, the drive shaft 116 can be modified to rotate via a magnetic coupling. For example, the lower section 130 of the drive shaft 116 can be sealed within the compartment 38 and have a magnetic coupling at one end. The magnetic drive can be disposed outside the housing 12 and magnetically coupled to the magnetic coupling. Then, as is known in the art, activation of the magnetic drive outside the housing 12 facilitates rotation of the drive shaft 116 / lower section 130 within the compartment 38. In this embodiment, the dynamic seal 122 can be eliminated and the risk of leakage or contamination is low.

[0092] The second end 120 of the drive shaft 116 extends toward the bottom end wall 16 and, in the depicted embodiment, can be rotatably supported on the bottom end wall 16. Figure 6, the bottom end wall 16 has an interior surface 132 and an opposing exterior surface 134. An optional stabilizing support 136 is mounted on the interior surface 132. The stabilizing support 136 has a top surface 138 on which a recess 140 is formed. The second end 120 of the drive shaft 116 terminates at a terminal 142, which is rotatably disposed within the recess 140. More specifically, in an exemplary embodiment, the stabilizing support 136 has a boundary surface 144 that defines the recess 140. An annular lip seal 146 projects radially inward from the boundary surface 144 so as to surround the terminal 142 of the drive shaft 116 and form a liquid-tight seal against the terminal. That is, the lip seal 146 seals against the drive shaft 116 to prevent the suspension in the compartment 38, or at least the cells therein, from entering the recess 140 and becoming lodged therein. However, the stabilizing support 136 still allows the drive shaft 116 to rotate relative to the stabilizing support 136. The boundary surface 144 may be in the form of a curved recess on which the terminal end 142 of the drive shaft 116 is directly supported. The terminal end 142 may also terminate in a rounded tip to help facilitate smooth rotation of the drive shaft 116 on the boundary surface 144.

[0093] In an exemplary embodiment, a sprinkler may be incorporated into the stabilizing support 136 to deliver bubbles into the compartment 38. For example, the stabilizing support 136 also has an interior surface 148 that at least partially defines a cavity 150. In one embodiment, the stabilizing support 136 may be welded or otherwise secured to the interior surface 132 of the bottom end wall 16 such that the cavity 150 is defined between the interior surface 132 of the bottom end wall 16 and the interior surface 148 of the stabilizing support 136. Alternatively, the stabilizing support 136 may also be formed with a bottom wall extending across the base of the stabilizing support 136 such that the cavity 150 is defined between the interior surface 132 and the bottom wall. A plurality of spaced apart gas openings 151 extend between the top surface 138 and the interior surface 148 of the stabilizing support 136. A gas line 152 is coupled to a port 153 on the bottom end wall 16 to communicate with the cavity 150. The gas line 152 is coupled to a gas source. During operation, gas is delivered from a gas source through gas line 152 into chamber 150. The gas then flows out through gas opening 151 to enter the suspension in compartment 38 in the form of bubbles. The bubbles flow upward through the suspension, and the gas then leaves compartment 38 through gas filter 100 (see FIG. Figure 5 ), as previously discussed. In alternative embodiments, as discussed below, other forms of gas sprayers may also be used. For example, the sprayer need not be incorporated into the stable support. Instead, the sprayer may be separate and detachable from the stable support 136.

[0094] Return to Figure 5, a plurality of spaced-apart mixing elements 154 are mounted on the drive shaft 116. In the depicted embodiment, three individual mixing elements 154 are shown disposed on the drive shaft 116. In alternative embodiments, it should be understood that the drive shaft 116 may be provided with at least one, two, three, four, or more individual mixing elements 154. The mixing elements 154 may include impellers, paddles, fins, protrusions, or other structures that promote mixing of the suspension within the compartment 38 when moved by the drive shaft 116 / drive motor 126.

[0095] During operation, the bioreactor 10 can initially be disposed in a horizontal orientation and operated in a static mode for a first time period without mixing. Once the cells reach a desired density, state, or meet some other predetermined condition, the bioreactor 10 can be rotated to a vertical orientation and operated in a dynamic mode, wherein mixing occurs for a second time period, a first second stage of cell growth, or other stages of cell processing. In an alternative exemplary embodiment, the bioreactor 10 can also be operated in a dynamic mode, wherein the drive shaft 116 and the mixing element 154 rotate while the bioreactor 10 is in a horizontal orientation and during a first time period, a first stage of cell growth, or other stages of cell processing.

[0096] More specifically, during use in one exemplary embodiment, a suspension is disposed within compartment 38 by dispensing cells, culture medium, nutrients, and other desired components into compartment 38, such as through ports 105 and / or 106. All or some of the components may be combined outside compartment 38 and then dispensed therein, or the components may be dispensed individually into compartment 38 to form a suspension therein. Figure 7 As shown, the bioreactor 10 (shown as bioreactor 10A) is positioned within a temperature and pressure controlled environment (such as within an incubator 160) before or after the suspension is formed within the compartment 38. The bioreactor 10 is placed in a horizontal orientation such that the first transfer opening 40 / gas permeable membrane 50 (see Figure 3 ) face downward. More specifically, Figure 3, the compartment 38 has a central longitudinal axis 162 that extends centrally through the compartment 38 so as to pass through the top end wall 14 and the bottom end wall 16. When in a horizontal orientation, the bioreactor 10 is arranged so that the front wall 28 is now the bottom surface and the axis 162 is oriented at any angle within a range between + / - 30 degrees, 20 degrees, 15 degrees, 10 degrees or 5 degrees. "Any angle" also includes the axis 162 being arranged horizontally. That is, when the horizontal orientation is used for operation of the bioreactor 10 in a static mode or a dynamic mode, the axis 162 is typically arranged horizontally. However, if the axis 162 is slightly angled relative to the horizontal plane, the bioreactor 10 can also be operated in a static mode or a dynamic mode. The bioreactor 10 can also be operated in a static mode, without mixing or without rotation / actuation of the drive shaft 116 / mixing element 154, while the bioreactor 10 is in a vertical orientation and is equipped with a gas permeable membrane (such as a gas permeable membrane 162) positioned at the bottom end wall 16. Fig. 9 Likewise, when in a vertical orientation, static mode operation may be utilized with forced gas exchange between the gas permeable membrane 172 and the gas phase of the bioreactor contents.

[0097] Placing the bioreactor 10 in a horizontal orientation with the first transfer opening 40 / gas permeable membrane 50 facing downward for operation during the static mode helps maximize the amount of cell suspension that contacts the gas permeable membrane 50, allowing for more accurate control of the gas content within the suspension. Additionally, with the first transfer opening 40 / gas permeable membrane 50 facing downward, the cells within the suspension settle under gravity to rest directly against or be disposed adjacent to the gas permeable membrane 50, thereby helping to ensure proper gas transfer to the cells. Furthermore, since the mixing element 154 and the sparger are typically not operated during the static mode when the bioreactor includes a gas permeable membrane 50, there is no need to consider optimal mixing efficiency or optimal sparging efficiency during static mode operation.

[0098] As discussed previously and Figure 7, multiple bioreactors 10A-10C can be simultaneously positioned within an incubator 160 (or other temperature and pressure controlled environment) to optimize cell production. In order to optimize the use of space within the incubator 160, a selected incubator, or multiple incubators, the bioreactors 10A-10C can be vertically stacked on top of each other when in a horizontal orientation using mounting portions 80 and 82, as previously discussed. For example, the bioreactors 10A-10C are shown as stacked within the incubator 160, wherein each bioreactor 10A-10C has the same configuration. Each of the multiple bioreactors 10A-10C has the same or substantially the same horizontal orientation and is vertically spaced apart from each other to allow gas to flow freely through the gas permeable membrane 50. Depending on the size of the bioreactors 10 and the size of the incubator 160, the bioreactors can be stacked at least 2, 3, 4, 5, 6, or 8 high, while at least 2, 4, 8, 12, 16, or 24 bioreactors 10 can be contained in the incubator 160 (or other temperature and pressure controlled environment) in a horizontal orientation. As previously discussed, the bioreactors 10 can also be stacked vertically in the incubator 160 or other temperature and pressure controlled environment when in a vertical orientation. Based on various sensor readings, the temperature and gas composition, concentration, and / or pressure within each bioreactor 10 can be selectively controlled by adjusting the temperature and gas composition, concentration, and / or pressure within the incubator 160 (or other temperature and controlled environment). For example, as previously mentioned, by controlling the oxygen concentration gradient between the suspension and the environment within the incubator 160, oxygen can reach the cells in the suspension through the gas permeable membrane 50 by gas diffusion. Similarly, controlling the CO2 concentration gradient between the suspension and the environment with the incubator 160 can be used to help control the pH of the suspension.

[0099] During cell expansion, when bioreactor 10 is in static mode, there is usually no mixing or interference of cell suspension. For example, drive motor 176 is usually not activated to promote the movement of mixing element 154. In addition, bioreactor 10 is usually not moved by tilting, shaking or rotating, nor is the cell therein disturbed by injecting liquid or gas into compartment 38. In addition, since gas transfer passes through gas permeable membrane 50, it is usually not necessary to spray gas into compartment 38. In some embodiments, gas can be made to flow through the gas phase of bioreactor contents instead of liquid to promote gas exchange without disturbing liquid. Cells are expanded in a first time period in static mode in bioreactor 10, and the first time period is usually at least 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, 120 hours or in the range between any two of the foregoing items. However, in some cases, it may be beneficial for the growth of cells to promote some intermittent disturbance (i.e., momentary movement) of the cells while the bioreactor 10 is operating in a dynamic mode by activating, actuating, or rotating the drive motor 176, drive shaft 116, and / or mixing element 154, or by shaking, rocking, tilting, or otherwise disturbing the bioreactor 10. When the bioreactor 10 is operating in a dynamic mode within the incubator 160, the disturbance of the cells may be achieved by short duration tilting, shaking, or rotation of the bioreactor 10, movement of the mixing element 154 within the bioreactor 10, injection of gas or liquid into the compartment 38, or other techniques. For example, the disturbance of the cells (such as by using one of the processes described above) may occur for a disturbance period of less than 15 minutes, 10 minutes, 5 minutes, 3 minutes, 2 minutes, 1 minute, 0.5 minutes, or 0.1 minutes, followed by a stasis period of at least 0.5 hours, 1 hour, 12 hours, 24 hours, or 48 hours, wherein no force is applied to disturb the suspension / cells, e.g., the bioreactor is operating in a static mode. When the bioreactor 10 is in the incubator 160 in alternating static mode and dynamic mode in a horizontal orientation ( Figure 7 ), in vertical orientation ( Fig. 9 The disturbance and stagnation periods may be repeated multiple times or continuously when operating in other angled orientations (as shown in FIG. 1 ) or at other angled orientations.

[0100] Subjecting the cells / suspension to a static mode (where the cell suspension is generally not mixed or disturbed during the first period of time) can be used to help promote treatment or post-treatment recovery of the cells. For example, in some applications, it may be necessary to treat the cells with a lentivirus. Lentiviruses can integrate large amounts of viral complementary DNA into the DNA of host cells and can effectively infect non-dividing cells, so they are one of the most effective gene delivery methods. In some cases, lentiviral transduction, transfection, application, integration or infiltration into cells may be more effective if done in a static mode (where no mixing or disturbance occurs).

[0101] In other applications, before or after the suspension is placed in the compartment 38 of the bioreactor 10, the cells / suspension may be electroporated. Electroporation is a microbiological technique in which an electric field is applied to the cells to increase the permeability of the cell membrane, thereby allowing chemicals, drugs, electrode arrays or DNA to be introduced into the cells. Electroporation destroys the cell membrane, and then generally requires direct static recovery with appropriate O2 and CO2 to maintain viability. Vigorous stirring of the cells immediately after electroporation may result in loss of viability. Thus, if initially processed in static mode after electroporation, the recovery of the cells may be improved.

[0102] Once a predetermined period of time has elapsed or it is detected that the cells have reached a predefined state or density or some other predefined condition has been met, the bioreactor 10 can be operated in a dynamic mode or a constant dynamic mode. For example, once the cells reach a predefined density, additional culture medium must be added to the compartment 38 to enable the cells to continue to expand. However, as the volume of the suspension in the compartment 38 increases, the volume of the suspension eventually reaches a point where gas transfer through the gas permeable membrane 50 alone is insufficient to maintain cell viability. When the bioreactor 10 is operated in a dynamic mode, gas can be sparged into the compartment 38 while the cells are mixed in the compartment, thereby increasing the transfer of gas to the cells. When operating in a dynamic mode, the bioreactor can be placed in a vertical or horizontal position.

[0103] In an exemplary embodiment, when in dynamic mode, bioreactor 10 is in a vertical orientation for accelerating cell growth after cell density reaches a predetermined threshold. Figure 3 , the horizontal bioreactor 10 is moved approximately 90 degrees so that the bottom end wall 16 is now the bottom surface and the first transfer opening 40 / gas permeable membrane 50 protrudes laterally. More specifically, the bioreactor 10 can be oriented so that the central axis 162 is oriented at any angle within a range between + / -30 degrees, 20 degrees, 15 degrees, 10 degrees or 5 degrees relative to the vertical direction. "Any angle" also includes the axis 162 being arranged vertically. That is, when operating in a dynamic mode (or static mode) in a vertical orientation, the axis 162 is generally arranged vertically. However, if the axis 162 is slightly angled relative to the vertical plane, the bioreactor 10 can also be operated in a dynamic mode. It should also be understood that, as described above, when moved to a vertical orientation, the bioreactor 10 can be operated in a static mode, and when in both a horizontal orientation and a vertical orientation, the bioreactor can be periodically moved between a static mode and a dynamic mode.

[0104] Once the bioreactor 10 is operated in the dynamic mode, mechanical mixing of the suspension can be initiated by activating the drive motor 126 to rotate the drive shaft 116 and thereby move the mixing element 154 within the suspension. In other exemplary embodiments, other types of mixing elements can be used, and other mixing techniques (such as tilting, shaking or rotating the bioreactor 10 or injecting gas or liquid into the compartment 38) can be used to mix the suspension. In addition, gas can be sprayed into the lower end of the compartment 38 during the dynamic mode, such as by a stable support 136 or a separate sprayer. Additional culture medium and / or nutrients are typically added to the compartment 38 before, during, or after the bioreactor 10 is operated in the dynamic mode (in a vertical orientation or a horizontal orientation).

[0105] The bioreactor 10 is specifically configured such that the width W is less than the height H, as previously described with respect to FIG. Figure 1 As discussed, in order to help optimize performance in static and dynamic modes and in horizontal and vertical orientations. For example, having a height H that is larger relative to the width W when in a horizontal orientation can maximize the surface area of ​​the gas permeable membrane 50 in contact with the suspension while minimizing the height of the suspension above the gas permeable membrane 50, thereby optimizing gas transfer across the gas permeable membrane 50 to the cells. In this way, gas exchange and cell expansion can be maximized or optimized when the reactor is in a static mode and a horizontal orientation. Subsequently, having a height H that is larger relative to the width W when in a vertical orientation and a dynamic mode keeps the suspension closer to the drive shaft 116 / mixing element 154, which helps to optimize both mechanical mixing efficiency and spraying efficiency within the compartment 38 at different levels of liquid volume.

[0106] When operating in dynamic mode in both the vertical and horizontal orientations, the bioreactor 10 can be maintained within an incubator 160 (or other temperature and pressure controlled environment) to help control temperature and other environmental conditions. However, more commonly, the bioreactor 10 is moved out of the incubator 160 when operating in dynamic mode and operating conditions are controlled separately. For example, referring to Figure 1 and Fig. 20, a heating jacket 420 can be disposed around the housing 12 to control the temperature of the suspension within the compartment 38 when the bioreactor 10 is operated in a dynamic mode outside the incubator 160. In an exemplary embodiment, the heating jacket 420 can include an insulating liner 422 that can be wrapped around the housing 12 into a loop and maintained in a desired configuration by a strip 424 that surrounds the exterior of the liner 422. An electric heating tape 426 or other heating element is disposed within the liner 422 or on its interior surface for controlling the temperature of the suspension within the compartment 38. As discussed below, other types of heating jackets or heating mechanisms can also be used to control the temperature of the suspension within the compartment 38. The bioreactor 10 is combined with the heating jacket 420 to form a bioreactor system.

[0107] With the bioreactor 10 in the dynamic mode, the cells are expanded in the compartment 38 for a second time period, which is typically at least 1 day, 2 days, 7 days, 14 days, 21 days, 60 days, or in a range between any two of the foregoing. In an exemplary embodiment, the spraying and mixing described above can be continuously operated during the second time period when the bioreactor 10 is in a vertical orientation. Once the predetermined time period has passed or it is detected that the cells in the compartment 38 have reached a predefined state or density or have met some other predefined condition, the suspension, culture medium and / or cells can be transferred out of the compartment 38 for subsequent use or further processing, such as further culture in a larger bioreactor.

[0108] Once the suspension or its desired portion has been removed from the compartment 38, the housing 12 and other parts thereof in contact with the suspension can be discarded as disposable items. In contrast, components such as the drive motor 126, the protective sleeve 128 and the sensor that do not directly contact the suspension can be removed and reused without any sterilization. In an exemplary method of use, in a static mode and / or a dynamic mode, the bioreactor 10 can be operated in a batch process, wherein the suspension is maintained in the compartment 38 during the process. In an alternative embodiment, in a static mode and / or a dynamic mode, a pipeline can be connected to the bioreactor 10 outside the compartment 38 to form a circulation loop so that the bioreactor 10 can be operated using a perfusion process. When the bioreactor 10 is used with or in a perfusion device, process or mode, cell expansion can occur in the bioreactor 10 for an extended period of time, such as at least 2 days, 5 days, 14 days, 21 days, 30 days or 60 days.

[0109] In the above process, the bioreactor 10 is used sequentially in both static mode and dynamic mode. However, in some cases, the bioreactor 10 can be used only in one mode or dynamic mode in static mode or dynamic mode and / or in alternating vertical orientation and horizontal orientation in either mode. That is, after the bioreactor 10 is operated only in static mode or dynamic mode, cells can be removed from the bioreactor 10, wherein the bioreactor 10 can be oriented in horizontal orientation or vertical orientation in either mode. In addition, when the bioreactor 10 is in vertical orientation or horizontal orientation and in dynamic mode, the bioreactor 10 can be arranged inside or outside the incubator (or other temperature and pressure controlled environment), or can be moved sequentially between them. Spraying is usually implemented when the bioreactor 10 is operated outside the incubator. However, depending on the conditions in the incubator, spraying may or may not be required when the bioreactor 10 is operated in dynamic mode in the incubator.

[0110] It should be understood that the bioreactor 10 may have a variety of alternative components that may be mixed and matched with other components and embodiments. Figure 8 , depending on factors such as the size of the housing 12, the amount of suspension being processed, and the configuration of the mixing elements, in some embodiments, the stabilizing support 136 may be eliminated. In this embodiment, the terminal end 142 of the drive shaft 116 may be freely suspended within the compartment 38 at a distance spaced apart from the bottom end wall 16. In addition, one or more of a variety of different types of sprinklers may be provided on the bottom end wall 16 for spraying gas into the compartment 38. The sprinklers may include porous frit sprinklers 166 or dome sprinklers 168, both of which are mounted on the bottom end wall 16 and protrude into the compartment 38. Alternatively, the sprinklers may include thin film sprinklers. The thin film sprinklers are flush with the end wall 16, so they do not impede the flow of fluid or the flow of cells or microorganisms within the compartment 38. Examples of various types of dome sprinklers and film sprinklers that may be used in the present disclosure are disclosed in U.S. Patent Nos. 8,603,805, 9,005,971, 9,259,692, 9,475,012, 9,682,353, 10,328,404, and 9,643,133, 10,350,554, 10,843,141, and U.S. Patent Publication No. 2021 / 0069654, which are incorporated herein by specific reference. Other conventional sprinklers may also be used. In another alternative embodiment, one or more of the individual sprinklers 166 and / or 168 may have a stabilizing support 136 incorporated therein, such as Figure 6 As shown, or it can be used independently of the stabilizing support 136.

[0111] Go to Fig. 9In another exemplary embodiment, the bottom end wall 16 may be formed with a second transfer opening 170 extending therethrough, which is at least partially covered by a gas permeable membrane 172. The gas permeable membrane 172 may have the same characteristics and composition as previously discussed with respect to the gas permeable membrane 50. A support frame 174 having the same construction, characteristics, and alternatives as the support frame 58 previously discussed may optionally be used to secure the gas permeable membrane 172 within the second transfer opening 170 or to an exterior surface of the bottom end wall 16 for sealingly closing the second transfer opening 170 while still allowing gas to pass through the gas permeable membrane 172. Alternatively, the gas permeable membrane 172 may be secured directly to the bottom end wall 16 without the use of a support frame 174. In other alternative embodiments, second transfer openings extending through the side wall 32 and / or the side wall 34 may also be formed. These openings may also be covered by corresponding gas permeable membranes with or without the use of a support frame to seal the transfer openings while still allowing gas to pass through the gas permeable membranes. These additional transfer openings with gas permeable membrane can be used to supplement the gas permeable membrane 50 in transferring gas to the suspension. In some embodiments, only the bottom end wall 16 has a gas permeable membrane mounted thereon, while the front wall 86 has no gas permeable membrane and no transfer openings and is made of exactly the same material as the side walls 32, 34. Figure 1 , Figure 8 and Fig. 9 The bioreactor 10 shown in the alternative exemplary embodiment (including the housing 12, the drive shaft 116 and its mixing element 154) can be used in dynamic and static modes, in both horizontal and vertical orientations, and inside and outside of an incubator or other temperature and pressure controlled environment.

[0112] The drive shaft 116 and the mixing element 154 may have a variety of different configurations. For example, Fig.10A drive shaft 180 is shown coupled to the drive motor 126. The drive shaft 180 includes a first drive shaft portion 182 having a spiral configuration and a second drive shaft portion 184 having a spiral configuration. The second drive shaft portion 184 is disposed laterally to the first drive shaft portion 182 and extends along the length of the first drive shaft portion 182. The drive shaft portions 182 and 184 are spaced apart and wound along their lengths to form a DNA spiral configuration. The first ends of the drive shaft portions 182 and 184 are connected together by a support 186A, while the second ends of the drive shaft portions 182 and 184 are connected together by a support 186B. An upper shaft portion 187 protrudes from the support 186A, passes through the top wall 14, and is coupled to the drive motor 126. The support 186B may be freely suspended or may have a lower shaft portion 190 protruding from it and connected to the stabilizing support 136. The drive shaft portions 182 and 184 are sufficiently rigid that they will deform with plastic deformation if twisted about their longitudinal axes under torsion beyond an angle of at least 45, 90, or 180 degrees. A mixing element 188 in the form of an impeller extends between the drive shaft portions 182 and 184. The mixing element 188 may also have other configurations that will promote mixing of the cell suspension when rotated by the drive shaft 180. Optionally, a spaced-apart brace 192 may also extend between the drive shaft portions 182 and 184 to help maintain stability therebetween.

[0113] Designing the drive shaft 180 to include drive shaft portions 182 and 184 having a spiral configuration helps match the mixing characteristics between the bioreactor 10 and a larger-sized bioreactor that uses an adjacent drive line that is twisted into a spiral configuration during the mixing process. An example of such a larger-scale bioreactor is disclosed in U.S. Patent No. 10,669,515. However, compared to a larger bioreactor in which the drive line is a flexible line, the drive shaft portions 182 and 184 are rigid members. Therefore, although not required, the use of the drive shaft 180 can help maintain uniform mixing of the cell suspension when the cell suspension is moved to a larger-sized bioreactor. However, in an alternative exemplary embodiment, the drive portions 182 and 184 can be formed by flexible tubing. That is, the bioreactor 10 and other bioreactors disclosed herein can be modified to incorporate a mixing assembly as disclosed in U.S. Patent No. 10,669,515, issued on June 2, 2020, which is incorporated herein by specific reference. The bioreactor 10 incorporating the drive shaft 180 and mixing element 188 can also be used in both dynamic and static modes, in both horizontal and vertical orientations, and inside and outside of an incubator or other temperature and pressure controlled environment.

[0114] Fig.11 and Fig.12Another exemplary embodiment of a bioreactor system 198 incorporating features of the present disclosure is depicted in . In general, the bioreactor system 198 includes a bioreactor 200 removably mounted on a heater support 202. Similar elements between the bioreactor 200 and the bioreactor 10 are identified by similar reference numerals. The bioreactor 200 includes a housing 12A having the same walls, faces, surfaces, and compartments as the housing 12, and thus the same elements between the housings 12 and 12A are identified by the same reference numerals. In addition, the disclosures, alternatives, and uses previously discussed with respect to the housing 12 also apply to the housing 12A. For example, the housing 12A includes a front wall 28, a rear wall 30, and side walls 32 and 34, each extending between an upper end 278 and an opposing lower end 279. The upper end 278 terminates at the top end wall 14, while the lower end 279 terminates at the bottom end wall 16 (see Fig.13 ). In this embodiment, all walls except the top wall 14 are shown as transparent or translucent, but may also be formed as opaque. The housing 12A defines a compartment 38. The first transfer opening 40 extends through the front wall 28 to communicate with the compartment 38. The gas permeable membrane 50 is used with or without the support frame 58 (see Figure 3 ) is fixed to the front wall 28 so as to cover and seal the first transfer opening 40. Again, the gas permeable membrane 50 and all prior disclosures with respect to the housing 12 being mounted on the front wall 28 also apply to the housing 12A.

[0115] Go to Fig.13 , the bottom end wall 16 of the housing 12A has an interior surface 204 and an opposing exterior surface 206. In the depicted embodiment, the bottom end wall 16 is shown as being integrally formed with the surrounding side wall 18. For example, the bottom end wall 16 and the surrounding side wall 18 can be molded as a single piece. However, in alternative embodiments, as discussed below, the bottom end wall 16 can be formed separately from the surrounding side wall 18, and the two elements are subsequently connected together. As will be discussed in more detail below, a pair of point sensors 220A and 220B are shown as being fixed on the interior surface 204 so as to face the exterior surface 206. The interior surface 204 slopes inwardly and downwardly to the discharge port 208. A tubular first rod 210 is disposed on the exterior surface 206 of the bottom end wall 16 and protrudes laterally so as to extend beyond the side wall 32. The first rod 210 defines a channel 212 that is in communication with the discharge port 208. An inlet port 214 is also formed on the interior surface 204 and is in communication with the compartment 38. A tubular second rod 216 is disposed on the exterior surface 206 of the bottom end wall 16 and projects laterally so as to also extend beyond the side wall 32. The second rod 216 defines a passage 218 that communicates with the inlet port 214. The rods 210 and 216 may project in parallel alignment.

[0116] Sprinkler 222 is also disposed on the interior surface 204 of bottom end wall 16. Although not required, in this embodiment, sprinkler 222 is annular and surrounds guide rod 223 that protrudes upward from interior surface 204. A tubular third rod 224 is disposed on the exterior surface 206 of bottom end wall 16 and protrudes laterally opposite rods 210 and 216 so as to also extend beyond side wall 34. Third rod 224 defines a passage 226 that communicates with sprinkler 222. Thus, gas passing through third rod 224 will pass through sprinkler 222 and enter chamber 38 in the form of bubbles. A tubular fourth rod 228 is disposed on the exterior surface 206 of bottom end wall 16 and extends along the length of bottom wall 16. Fourth rod 228 defines a passage 230 that is not in communication with compartment 38, but is in communication with the environment through opening 232. As will be discussed in more detail below, passage 230 is configured to receive a temperature sensor and may protrude parallel to third rod 224. In the depicted embodiment, rods 210, 216, 224, and 228 are integrally formed with bottom end wall 16 so as to form a single, unitary piece with bottom end wall 16, rather than being separately connected to the bottom end wall. For example, rods 210, 216, 224, and 228 may be integrally molded with bottom end wall 16. In alternative embodiments, one or more of rods 210, 216, 224, and 228 may be separately connected to bottom end wall 16.

[0117] like Fig.11 and Fig.12 , tube 234 is coupled to first rod 210 and can be used to drain, sample, or otherwise remove all or a portion of the cell suspension from compartment 38. Tube 236 is coupled to second rod 216 and can be used to deliver culture medium, nutrients, cell suspension, and / or components thereof to compartment 38. Tube 238 is coupled to third rod 224 and can be used to deliver gas to sparger 222.

[0118] Go to Fig.14 and Fig.15 , the top end wall 14 is again configured for mounting to an upper end 278 of the surrounding side wall 18, as previously discussed. The top end wall 14 includes an exterior surface 264 and an opposing interior surface 265, and more specifically includes a top panel 266 and a mounting lip 274 protruding therefrom, as previously discussed. Ports 240, 242, and 244 pass through the top end wall 14 and communicate with the compartment 38. One or more of the ports 240, 242, and 244 may optionally receive a sensor, such as the sensor 94 or 96 discussed previously. When not in use, the ports 240, 242, and / or 244 may each be sealed by a plug 246. Two spaced-apart optional tubular brackets 115 protrude upwardly from the exterior surface 264 of the top end wall 14, each of which is configured to receive a hanger 112 (see Fig.11), as previously discussed. On top end wall 14, first pair of ports 105A and 106A and second pair of ports 105B and 106B are also formed to pass therethrough. Ports 105 and 106 provide the optional entrance leading to compartment 36, for delivering and / or removing cell suspension or its component. Each of ports 105 and 106 may comprise an upper rod 250 protruding upwards from the external surface 264 of top end wall 14 and a lower rod 252 protruding downwards from the internal surface 265 of top end wall 14. Rod 250 and 252 make it possible to be easily connected with pipe. For example, pipe can be connected with each rod 250 to extend to compartment 38 outside, and independent pipe can be connected with each rod 252 and extend in compartment 38. However, rod 250 and 252 are optional, and other mechanisms can be used to couple pipe to ports 105 and 106.

[0119] A port 102 extending through the top end wall 14 can be connected to a tube 104 and a gas filter 100 (see Fig.11 ) is coupled, as previously discussed, to allow gas to escape from compartment 38. A dynamic seal 122 is centrally formed on top end wall 14, the dynamic seal having at least a portion of drive shaft 116 extending therethrough. Figure 5 , the upper end of the drive shaft 116 is coupled to the drive motor 126. The lower end of the drive shaft 116 protrudes into the compartment 38 and has a mixing element 158 ​​disposed therein. During use, the drive motor 126 facilitates the rotation of the drive shaft 116, which moves the mixing element 158 ​​within the compartment 36 for mixing the cell suspension. As previously discussed with respect to the bioreactor 10, other types of mixing systems (such as magnetic mixing systems) can be used on the bioreactor 200.

[0120] Return to Fig.14 and Fig.15 A pair of spaced-apart tube clips 256A and 256B are mounted on the top end wall 14 so as to protrude beyond the side wall 32, while a pair of spaced-apart tube clips 258A and 258B are mounted on the top end wall 14 so as to protrude beyond the side wall 34. Each tube clip includes a C-shaped or J-shaped slot or hook 260 that is configured to removably receive and retain a tube, such as Fig.11 Tubes 234 and 236 are shown. Thus, the tube clips can be used to organize, support and / or straighten tubes used with the bioreactor 200. The tube clips can also be placed in other locations and have other shapes, including open rectangles, open ovals, and similar surrounding structures.

[0121] Go to Fig.16 and Fig.17The heater support 202 includes a body 280 having a top wall 282, an opposing bottom wall 284, and a peripheral wall 286 extending therebetween. A plurality of spaced apart support legs 289 project downwardly from the bottom wall 284 of the body 280, on which the heater support 202 rests. A recess 288 is recessed in the top wall 282 so as to be at least partially surrounded by the peripheral wall 286. As discussed below, the recess 288 is configured to receive the lower end of the housing 12A. The bottom wall 284 has an interior surface 285 that forms a floor of the recess 288. Fig.11 As shown, one or more heating elements 291 are disposed within the body 280. More specifically, the one or more heating elements 291 typically extend laterally within the bottom wall 284 below the recess 288, but may also extend into the peripheral wall 286. The one or more heating elements 291 are typically electrical heating elements that can be powered by an electrical socket 293 (see FIG. 2 ) formed on the body 280. Fig.12 ) is powered. The controller 295 can be used to regulate the operation of one or more heating elements 291 (e.g., turning them on and off and controlling their temperature). The controller 295 can be arranged to be spaced apart from the heater support 202 and electrically connected to the heater support through wired or wireless communication. Alternatively, the controller 295 can be mounted directly on the body 280. The body 280, or at least the portion thereof defining the recess 288, is typically made of metal. However, other materials that effectively transfer heat can also be used.

[0122] The peripheral wall 286 of the heater support 202 includes a front wall 290, a rear wall 292, and a pair of opposing side walls 294 and 296 extending therebetween, each of which partially defines the recess 288. Fig.11 and Fig.16 As shown, a first slot 298 and a spaced apart second slot 300 extend through the side wall 294 to communicate with the recess 288. The slots 298 and 300 are generally linear and extend in parallel alignment through the top wall 282 and project downwardly toward the bottom wall 284. A first recess 302 and a second recess 304 are recessed into the interior surface 285 of the bottom wall 284 and intersect the first slot 298 and the second slot 300, respectively. The slots 298 and 300 may also be linear and extend in parallel alignment from the side wall 294 toward the side wall 296.

[0123] Go to Fig.12 and Fig.16, a third slot 306 passes through the side wall 296 so as to communicate with the recess 288. The third slot 306 is linear and extends through the top wall 282 and protrudes downward toward the bottom wall 284. A third recess 308 is recessed in the interior surface 285 of the bottom wall 284 and intersects with the third slot 306. The third slot 306 is linear and extends from the side wall 296 toward the side wall 294. A fourth recess 310 is also recessed in the interior surface 285 of the bottom wall 284. However, the fourth recess 310 does not intersect with the slot. Instead, the fourth recess 310 communicates with an opening 312 that passes through the side wall 296 and is completely surrounded by the side wall 296. The fourth recess 310 is also linear and may extend parallel to the third recess 308.

[0124] The recess 288 is configured to receive the lower end 279 of the housing 12A / bioreactor 200 so that the housing 12A / bioreactor 200 is securely supported in a vertical orientation, such as Fig.11 and Fig.12 Specifically, refer to Fig.11 , Fig.13 and Fig.16 When it is desired to place the housing 12A / bioreactor 200 in a vertical orientation (such as when used in a dynamic mode), the lower end 279 of the housing 12A / bioreactor 200 is lowered into the recess 288 so that the first rod 210, the second rod 216, and the third rod 224 of the housing 12A (where each rod typically has a tube coupled thereto) pass downwardly through and protrude from the first slot 298, the second slot 300, and the third slot 306 of the heater support 202, respectively. Subsequently, when the lower end 279 of the housing 12A rests on the interior surface 285, the first rod 210, the second rod 216, the third rod 224, and the fourth rod 228 of the housing 12A are received within the first recess 302, the second recess 304, the third recess 308, and the fourth recess 310 of the heater support 202, respectively. That is, in one exemplary embodiment, the interior surface 285 of the heater support 202 has a complementary profile to the exterior surface 206 of the bottom end wall 16, so that a relatively close tolerance fit is formed therebetween. This complementary fit helps stabilize the housing 12A / bioreactor 200 on the heater support 202, and helps facilitate heat transfer between the heater support 202 and the housing 12A / bioreactor 200, as discussed below. In addition, the alignment between the various rods, slots, and recesses helps facilitate easy and correct insertion of the housing 12A / bioreactor 200 into the recess 288. It should also be understood that the bioreactor 200 can be operated in a static mode when mounted on the heater support 202.

[0125] When the fourth rod 228 is received in the fourth recess 310, the channel 230 in the fourth rod 228 is aligned with the opening 312 that passes through the side wall 296 and enters the fourth recess 310. Through this alignment, the temperature sensor 316 can pass through the opening 312 and enter the channel 230 of the fourth rod 228. The temperature sensor 316 detects the temperature of the cell suspension in the heater support 202 and / or the compartment 38. The temperature detected by the temperature sensor 316 can be used to control the operation of the heater support 202, that is, to increase or decrease its temperature so that the cell suspension is maintained at a desired temperature. The temperature sensor 316 can be electrically connected to the controller 295 to facilitate automatic adjustment of the temperature. Alternatively, the data received from the temperature sensor 316 can be used to facilitate manual adjustment of the controller 295. In an exemplary embodiment, the temperature sensor 316 can be a resistance temperature detector (RTD). However, other types of temperature sensors can also be used.

[0126] By using the temperature sensor 316 in the 4th bar 228, the accurate measurement of the temperature of the cell suspension in the compartment 38 can be obtained without the need for the temperature sensor 316 to directly contact the cell suspension. Thus, the risk of contaminating the cell suspension is less, and the temperature sensor 316 is reusable without any cleaning or sterilization. In addition, in some embodiments, the use of the temperature sensor 316 in the 4th bar 228 can eliminate the need for the temperature sensor to be placed in the compartment 38 through the top wall 14 downwards. That is, the sensor (such as sensor 94 and 96) of the port 240,242 and 244 passing through the top wall 14 downwards does not need to be a temperature sensor, thereby leaving room for other sensors or enabling to delete one of the ports 240,242 and 244 to simplify production. In other embodiments, when using the temperature sensor that passes through the top wall 14 downwards and enters the compartment 38, the 4th bar 228, the 4th recess 310 and the temperature sensor 316 can be eliminated.

[0127] In alternative embodiments, the arrangement of the various slots and recesses on the heater support can be adjusted. For example, all three slots 298, 300 and 306 and the opening 312 can be formed on either of the sidewalls 294 and 296, rather than having slots 298 and 300 on the sidewall 294 and slots 306 and openings 312 on the sidewall 296. In other embodiments, any combination of slots 298, 300 and 306 and openings 312 can be formed on the sidewalls 294 and 296. It should be understood that when the slots 298, 300 and 306 and the opening 312 are moved to different sidewalls 294 and 296, the corresponding recesses 302, 304, 308 and 310 are also moved so as to be aligned accordingly therewith. Similarly, the positioning of the rods 210, 216, 224 and 228 will be modified to be consistent with the changes in the arrangement of the slots and recesses on the heater support 202. In other embodiments, any one or combination of the first rod 210, the second rod 216 and / or the third rod 224 and the corresponding first recess 302, the second recess 304 and / or the third recess 308 having corresponding slots of the housing 12A can be eliminated by passing the corresponding inlet pipe, outlet pipe and / or spray pipe downward through the top end wall 14 and into the compartment 38.

[0128] refer to Fig.13 , Fig.14 and Fig.17In one embodiment of the present disclosure, an optical sensor system can be used to detect O2, pH and / or CO2 within a cell culture. The optical sensor system typically includes an emitter / detector 320A electrically coupled to a controller 322 and a physicochemical reaction point sensor 220A or other appropriate sensor. The point sensor 220A is mounted on an interior surface of the housing 12A so that it will contact the cell suspension within the compartment 38. In the depicted embodiment, the point sensor 220A is mounted on the interior surface of the bottom end wall 16. However, in other embodiments, the point sensor 220A may also be mounted on the interior surface of the side wall 18. The emitter / detector 320A is positioned outside the housing 12A, but adjacent to and aligned with the point sensor 220A. In the depicted embodiment, the hole 324A extends through the bottom wall 284 of the heater support 202 so as to communicate with the recess 288. The hole 324A and the point sensor 220A are positioned so that when the emitter / detector 320A is fixed in the hole 324A, the front surface 326 of the emitter / detector 320A is arranged adjacent to the point sensor 220A and vertically aligned with it. When used in conjunction with the controller 322, the emitter / detector 320A is configured to emit light through the front 326 and emit it to the point sensor 220A. For example, the emitter / detector 320A may include an LED for emitting light. Then, the point sensor 220A emits fluorescence in response to light stimulation and the conditions in the cell suspension detected by the emitter / detector 320A. The detected information is then transmitted to the controller 322, and the controller can determine the pH, O2 or CO2 in the cell suspension according to the optical sensor system used. Such optical sensor systems can be purchased from PreSen Precision Sensing GmbH in Germany.

[0129] If desired, one, two, or three separate point sensors may be mounted on the interior surface of housing 12A and may each be used with a corresponding emitter / detector that is electrically coupled to controller 322. Each of the separate point sensors and emitters / detectors may be used to measure a single one of pH, O2, or CO2. For example, Fig.16 and Fig.17The use of emitters / detectors 320A and 320B is shown, which operate with separate point sensors 220A and 220B disposed on the housing 12A and each measure a different one of pH, O2 or CO2. By using point sensors 220, the previous sensors previously described for protruding downward through the top wall 14 and into the compartment 38, such as sensors 94 and 96 to measure pH, O2 or CO2, can be eliminated. This helps to simplify the design and production of the housing 12A. In addition, the relatively inexpensive point sensor 220 can be discarded after a single use, while the emitter / detector 320 and controller 322, which do not contact the cell suspension, can be reused without the need for cleaning or sterilization.

[0130] The bioreactor 200 can be used in substantially the same manner as the previously discussed bioreactor 10. For example, the initial bioreactor 200 separated from the heater support 202 can be positioned in a horizontal orientation within the incubator 160 (see Figure 7 ) in order to operate in a static mode. All previous discussions, methods and alternatives for operating the bioreactor 10 in a static mode are also applicable to operating the bioreactor 200 in a static mode. Therefore, such previous disclosures are incorporated with respect to the bioreactor 200, but are not repeated. Although the bioreactor 200 does not show a first mounting portion 80 and a second mounting portion 82 disposed on the housing 12A, the same mounting portions 80 and 82 and the alternatives discussed therewith may also be used on the housing 12A to facilitate stacking of the bioreactor 200 within the incubator 160 when in a horizontal orientation. However, the positioning of the mounting portions 80 and 82 at the lower end 279 of the housing 12A may need to be adjusted upwardly toward the upper end 278 so that the lower end 279 can be inserted into the recess 288 of the heater support 202 when in a vertical orientation / dynamic mode. Alternatively, the mounting portions 80 and 82 may be placed at the same location, but removed before being inserted into the recess 288. In addition, the mounting portions 80 and 82 are not required when stacking is not implemented.

[0131] Once a predetermined period of time has elapsed or it is detected that the cells have reached a predefined state or density or some other predefined condition has been met, the bioreactor 200 can be operated in a dynamic mode in a vertical orientation or a horizontal orientation inside or outside of the incubator 160. In an exemplary embodiment, the bioreactor 200 is operated in a dynamic mode in a vertical orientation. Likewise, all previous discussions, methods, and alternatives regarding moving and operating the bioreactor 10 in a dynamic mode (vertical orientation or horizontal orientation) also apply to the bioreactor 200. Therefore, such previous disclosures are incorporated herein with respect to the bioreactor 200, but are not repeated. However, with the use of Fig. 20In an alternative method of use, the lower end 279 of the bioreactor 200 can be fitted into the recess 288 of the heater support 202. The heater support 202 can then be used to control the temperature of the cell suspension in the compartment 38. In addition, as discussed above, the temperature sensor 316 and the optical sensor system ( Fig.13 ) may optionally be used to monitor the temperature, pH, O2 and / or CO2 of the cell suspension within compartment 38.

[0132] Fig.18 2B, which may be used as a replacement for housing 12A in bioreactor 200. Similar elements between housings 12A and 12B are identified by similar reference characters. Housing 12B is identical to housing 12A, except that housing 12B includes bottom end wall 16A, which is now formed separately from side wall 18 and is designed to be subsequently attached to side wall 18 during assembly. Specifically, housing 12B includes side wall 18 extending between upper end 278 and lower end 279. Upper end 278 has an opening 330 formed therein, which is surrounded by upper edge 277. Upper edge 277 engages top end wall 14, as previously discussed.

[0133] The lower end 279 now has an opening 332 formed therein, which is surrounded by a lower edge 333. The lower edge 333 / lower end 279 engages with the bottom end wall 16A in substantially the same manner as the upper edge 277 / upper end 278 engages with the top end wall 14. For example, the bottom end wall 16A includes a bottom panel 334. The bottom panel 334 includes an interior surface 204 and an exterior surface 206, which have the same construction, the same components, and the same alternatives as the bottom end wall 16 of the bioreactor 200 discussed previously. The interior surface 204 and the exterior surface 206 each extend to a peripheral edge 336. A mounting lip 338 protrudes upward from the interior surface 204 so as to be slightly embedded from the peripheral edge 336 and form a continuous ring. During assembly, the mounting lip 338 slides into the opening 332 formed at the lower end 279 of the side wall 18, while the interior surface 204 of the bottom panel 334 is located on the lower edge 333 surrounding the side wall 18. The mounting lip 338 is sealed to the interior surface of the surrounding side wall 18 to close the opening 332. The sealing joint can be achieved by welding, adhesives, press-fit connections, use of sealing rings or other conventional techniques. The above-described configuration of the bottom end wall 16 simplifies the production of both the bottom end wall 16A and the surrounding side wall 18, and provides easy engagement therebetween. However, it should be understood that the bottom end wall 16A may also have a variety of other configurations for mounting at the lower end 279 of the surrounding side wall 18.

[0134] Fig.19Another exemplary embodiment of a bioreactor system 350 incorporating features of the present disclosure is depicted in . Similar elements between the bioreactor system 350 and the bioreactor system 198 are identified by similar reference numerals. Generally speaking, the bioreactor system 350 includes a bioreactor 352 that is removably coupled to the heater support 202 discussed previously. The bioreactor 352 includes a bag assembly 354 that is supported within a support housing 356 and optionally operates with a base plate 358.

[0135] The bag assembly 354 includes a flexible collapsible bag 360 having a surrounding sidewall 362 extending from an upper end 364 to an opposite lower end 366. The upper end 364 terminates at a top end wall 368, while the lower end 366 terminates at a bottom end wall 370. The bag 36 also has an interior surface 372 defining a compartment 374. The compartment 374 is configured to hold a fluid. The bag 360 can be formed so that the compartment 374 can have any alternative volume as previously discussed with respect to the bioreactor 10. Similarly, the bag 360 can have the same alternative dimensions as previously discussed with respect to the bioreactor 10, for example, a height to width / diameter ratio. The bag 360 is composed of one or more flexible, water-impermeable polymer film (such as low-density polyethylene or FEP) sheets. The thickness of the polymer film can be at least or less than 0.02 mm, 0.05 mm, 0.1 mm, 0.2 mm, 0.5 mm, 1 mm, 2 mm, 3 mm, or in the range between any two of the aforementioned thicknesses. Other thicknesses can also be used. The film is flexible enough to be rolled into a tube without plastic deformation and can be folded at an angle of at least 90°, 180°, 270° or 360° without plastic deformation.

[0136] The film may be composed of a single layer of material or may include two or more layers, which are sealed together or separated to form a double-walled container. In the case where the layers are sealed together, the material may include a laminated or extruded material. The laminated material includes two or more layers formed separately, which are subsequently fixed together by an adhesive. An example of an extruded material that can be used in the present invention is the Thermo Scientific CX3-9 film purchased from Thermo Fisher Scientific. The Thermo Scientific CX3-9 film is a three-layer, 9-mil cast film produced in a cGMP facility. The outer layer is a polyester elastomer coextruded with an ultra-low density polyethylene product contact layer. Another example of an extruded material that can be used in the present invention is the ThermoScientific CX5-14 cast film also purchased from Thermo Fisher Scientific. The Thermo Scientific CX5-14 cast film includes a polyester elastomer outer layer, an ultra-low density polyethylene contact layer, and an EVOH barrier layer disposed therebetween.

[0137] The material can be approved for direct contact with living cells and can maintain the solution aseptic. In this embodiment, the material can also be sterilized such as by ionizing radiation. Examples of materials that can be used in different situations are disclosed in the U.S. Patent 6,083,587 issued on July 4, 2000 and the U.S. Patent Publication No. US2003-0077466A1 disclosed on April 24, 2003, which are hereby incorporated by reference in a specific manner.

[0138] In one embodiment, bag 360 may include a two-dimensional pillow bag. In another embodiment, bag 360 may be formed by a continuous tubular extrudate of a polymeric material cut to length. Each end may be sutured closed, or a panel may be sealed over an open end to form a three-dimensional bag. The three-dimensional bag has not only an annular sidewall, but also a two-dimensional top end wall and a two-dimensional bottom end wall. The three-dimensional container may include a plurality of discrete panels, typically three or more, and more typically four to six. Each panel is substantially the same and includes a portion of the sidewall, top end wall, and bottom end wall of the container. The corresponding peripheral edges of each panel are sutured together. The gap is typically formed using methods known in the art such as heat, RF energy, acoustic energy, or other sealing energy.

[0139] In alternative embodiments, the panels may be formed in a variety of different patterns. Further disclosure regarding one method of making a three-dimensional bag is disclosed in US Patent Publication No. US2002-0131654A1 published on September 19, 2002, which is incorporated herein by specific reference in its entirety.

[0140] Optionally, many of the same elements as previously discussed are provided on the top wall 14 of the bioreactor 200 are provided on the top wall 368. Specifically, optional ports 240 and 242 are provided on the top wall 368 that pass through the top wall 368 and communicate with the compartment 374. One or more of the ports 240 and 242 can optionally receive a sensor, such as the sensors 94 and 96 discussed previously. When not in use, the ports 240 and / or 242 can be sealed by a plug 246 (see Fig.14 ). A first pair of ports 105A and 106A and a second pair of ports 105B and 106B are also formed on the top wall 368 to pass therethrough. Ports 105 and 106 provide optional access to compartment 374 for delivering a cell suspension or its components to compartment 374 and / or removing a cell suspension or its components from the compartment. Ports 105 and 106 can be coupled to a tube extending outside of compartment 374 and a tube protruding into compartment 374. Port 102 extends through top wall 368 and can be coupled to tube 104 and gas filter 100 (see Fig.11 ) connection, as previously discussed. A dynamic seal 122 is centrally formed on the top end wall 368, the dynamic seal having at least a portion of the drive shaft 116 extending therethrough. As previously discussed and Figure 5 , the upper end of the drive shaft 116 is coupled to the drive motor 126. The lower end of the drive shaft 116 protrudes into the compartment 374 and has a mixing element 158 ​​disposed therein. During use, the drive motor 126 promotes the rotation of the drive shaft 116, which moves the mixing element 158 ​​within the compartment 374 for mixing the cell suspension therein. As previously discussed with respect to the bioreactor 10, other types of mixing systems (such as magnetic mixing systems) can be used on the bioreactor 352.

[0141] Tubes 234 and 236 are coupled to and extend from bottom end wall 370, and again these tubes may optionally be used to deliver a cell suspension or its components to and / or remove a cell suspension or its components from compartment 374. Tube 238 also extends from bottom end wall 370 and may be coupled to sparger 222 (see FIG. 1 ) disposed within compartment 374. Fig.13 ) connection. Tube 238 is used to deliver gas to sparger 222. Each of tubes 234, 236 and 238 can be connected to a port mounted on bottom end wall 370 and communicating with compartment 374.

[0142] The surrounding sidewall 362 may include a front wall 376, an opposing rear wall 378, and opposing side walls 380 and 382 extending therebetween. The first transfer opening 40 extends through the surrounding sidewall 362 / front wall 376 to communicate with the compartment 374. In the same manner as the previously discussed bioreactor 10, the bioreactor may be configured with or without the support frame 58 (see Figure 3 ), the gas permeable membrane 50 is secured to the surrounding sidewall 362 / front wall 376 so as to cover and seal the first transfer opening 40. Likewise, all prior disclosures regarding the gas permeable membrane 50 and mounting on the front wall 28 relative to the housing 12 also apply to the bag assembly 354. In an alternative embodiment, the bag 360 may be formed of a gas permeable membrane, as discussed herein, so as to eliminate the need to form the first transfer opening 40 or mount the gas permeable membrane 50 thereon.

[0143] The support housing 356 includes a support wall 386 that surrounds the chamber 387 and extends between an upper end 388 and an opposite lower end 390. The upper end 388 has an opening 392 formed therein that communicates with the chamber 387 and is surrounded by a top lip 394. The lower end 390 has an opening 396 formed therein that communicates with the chamber 387 and is surrounded by a bottom lip 398. Optionally, the tube clips 256 and 258 are secured to the top lip 394. In addition, spaced-apart tubular brackets 115 may be optionally secured to the top lip 394, each tubular bracket being configured to receive a hanger 112 (see FIG. 1 ). Fig.11 ), as discussed previously.

[0144] The chamber 387 is sized and configured to receive and support the bag assembly 354 therein. An inlet opening 400 of comparable size to the transfer opening 40 and / or the gas permeable membrane 50 extends laterally through the support wall 386. Specifically, the inlet opening 400 is sized and positioned on the support wall 386 so that when the bag assembly 354 is received within the chamber 387, the gas permeable membrane 50 is aligned with the inlet opening 400 and can breathe through the inlet opening. To help support the bag assembly 354 / gas permeable membrane 50 so that it does not excessively protrude or protrude outside of the inlet opening 400 while still allowing the gas permeable membrane 50 to breathe, a support structure 402 can be formed to extend across the inlet opening 400, directly supporting the gas permeable membrane 50. The support structure 402 can include a plurality of elongated rods extending through the inlet opening 400 laterally, vertically, or at an angle. The plurality of rods can be spaced apart or partially spaced apart, for example, they can intersect or interconnect at spaced apart locations. In one exemplary embodiment, the support structure 402 may be in the form of a grid structure. As an alternative to incorporating a support structure 402, the inlet opening 400 may be formed as a plurality of spaced-apart inlet openings 400, each inlet opening being in communication with the gas permeable membrane 50. In this embodiment, the remaining portion of the support wall 386 between the plurality of inlet openings 400 will provide support for the gas permeable membrane 50. However, in any design, the inlet openings need to be large enough to allow the gas permeable membrane 50 to breathe properly.

[0145] The bottom plate 358 can be used to help support the bag assembly 354 within the chamber 387 of the support housing 356. The bottom plate 358 can be permanently fixed to the lower end 390 of the support housing 356 or can be removably fixed to the lower end 390 so as to partially cover the opening 396. In other embodiments, the bottom plate 358 does not need to be connected to the lower end 390, but can simply be aligned with the opening 396. In other embodiments, the bottom plate 358 can be eliminated. The bottom plate 358 has a top surface 404 and an opposing bottom surface. Optionally, the openings 408A and 408B can extend through the bottom plate 358 to align with the emitters / detectors 320A and 320B disposed on the heater support 202. In this embodiment, the point sensors 220A and 220B (see Fig.13 ) can be mounted on the interior surface 372 of the bottom end wall 370 of the bag 360 for use with the emitter / detectors 320A and 320B, as previously discussed. Slots 410 and 412 also extend through the bottom plate 358, which align with the recesses 302 and 304 on the heater bracket 202 and allow the tubes 234 and 236 of the bag assembly 354 to pass through. Similarly, slot 414 extends through the bottom plate 358, aligned with the recess 308 of the heater bracket 202 (see Fig.16 ) to allow the tube 238 of the bag assembly 354 to pass through. Finally, the narrow slot 416 passes through the bottom plate 358 and aligns with the recess 310 (see Fig.16 ) to allow communication between the temperature sensor 316 and the bag assembly 354 when the temperature sensor 316 is received within the recess 310.

[0146] The bioreactor system 350 is capable of operating in both static and dynamic modes, and in both modes in a horizontal orientation, a vertical orientation, or an angled orientation, in substantially the same manner as previously discussed with respect to the bioreactor 200 and the bioreactor 10. The only difference is that during both methods of operation, the bag assembly 354 remains within the support housing 356. For example, the bioreactor 352 with the bag assembly 354 received within the support housing 356 (but separated from the heater support 202) is initially positioned within the incubator 160 (see Figure 6) to operate in a static mode (in a horizontal orientation, a vertical orientation, or an angled orientation). In an exemplary embodiment, when in the static mode (without mixing), the bioreactor system 350 is horizontally oriented with the membrane 50 facing downward, and when in the dynamic mode (with mixing), the bioreactor system is vertically oriented with a sprinkler (not shown) operating. All previous discussions, methods, and alternatives for operating the bioreactor 10 in the static mode also apply to operating the bioreactor 352 in the static mode. Therefore, such previous disclosures are incorporated with respect to the bioreactor 352, but are not repeated. Although the bioreactor 352 does not show a first mounting portion 80 and a second mounting portion 82 disposed on the support housing 356, the same mounting portions 80 and 82 and the alternatives discussed therewith may be used on the housing 356 to facilitate, for example, stacking of the bioreactor 352 within the incubator 160 when in a horizontal orientation. However, the positioning of the mounts 80 and 82 at the lower end 390 of the support housing 356 may need to be adjusted upwardly toward the upper end 388 so that the lower end 390 can be inserted into the recess 288 of the heater support 202 when moved to a vertical orientation (such as when operating in a dynamic mode). Alternatively, the mounts 80 and 82 may be placed at the same location but removed prior to insertion into the recess 288. Furthermore, where stacking is not implemented, the mounts 80 and 82 are not required.

[0147] After the initial period of static mode cell expansion, once a predetermined period of time has elapsed or it is detected that the cells have reached a predefined state or density or some other predefined condition has been met, the bioreactor 352 can be switched to and operated in the dynamic mode. Likewise, all previous discussions, methods, and alternatives regarding moving the bioreactors 10 and 200 to the dynamic mode also apply to the bioreactor 352. Therefore, such previous disclosures are incorporated with respect to the bioreactor 352 but are not repeated.

[0148] It is also important to note that each of the bioreactor systems 198 and 350 and their bioreactors, as well as other bioreactors disclosed herein, can be operated in both a vertical and horizontal orientation, inside or outside a temperature and pressure controlled environment (such as an incubator 160), can be operated in a static mode or a dynamic mode, inside or outside a temperature and pressure controlled environment (such as an incubator 160), can be operated in a static mode when in both a vertical and horizontal orientation, can be operated in a dynamic mode when in both a vertical and horizontal orientation, and can periodically switch between a static mode and a dynamic mode when in both a vertical and horizontal orientation.

[0149] Fig.21 is a perspective view of a bioreactor 510 according to an example embodiment and incorporating features of the present disclosure. Fig.21 The bioreactor 510 depicted in FIG. Figures 1 to 3 and contains many of the same features and components. However, Fig.21 The bioreactor 510 does not have a structure provided on the housing 12 or at the first transfer opening 40 ( Figures 1 to 3 510). The bioreactor 510 includes a housing 512 having a top end wall 514, an opposite bottom end wall 516, and a surrounding side wall 518 extending therebetween. The housing 512 may be made of a transparent material to allow visual inspection of the contents and components within the bioreactor 510. The surrounding side wall 518 has a front wall 520 including a front face, an opposite rear wall 522 including a rear face, and opposite side walls 524 and 526 extending between the top end wall 514 and the bottom end wall 516, respectively. The side walls 524 and 526 also include side faces. In the depicted embodiment, the surrounding side wall 518 has a rectangular or square transverse cross section. However, in other embodiments, the surrounding side wall 518 may have other transverse cross-sectional configurations, such as circular, elliptical, or polygonal. In the exemplary embodiment, the housing 512 has a cubic shape. The curvature of each corner of the housing 512 can be made with a radius of curvature or a sharp or rounded edge angled in a manner to prevent biological components and cells from accumulating in the corners of the reactor 510. The curvature of each corner of the housing 512 can also be different to form a mistake-proofing feature to ensure the correct orientation of the bioreactor 510.

[0150] Shell 512 has an inner surface 536 defining compartment 538. The volume of compartment 538 can be at least or less than 50 milliliters, 100 milliliters, 250 milliliters, 500 milliliters, 1 liter, 5 liters, 10 liters, 20 liters, 30 liters, 40 liters, 50 liters or in the range between any two of the foregoing. For example, compartment 538 generally has a volume in the range between 250 milliliters and 50 liters, wherein between 1 liter and 20 liters or between 1 liter and 10 liters is more common. Other volumes can also be used. In one embodiment, shell 512 and its wall are made of gas and liquid impermeable material (such as polymeric material and / or biocompatible material). In other examples, the compartment can have a volume greater than 50 liters.

[0151] In addition, the shell 512 and its walls 514, 516 are generally rigid. For example, in an exemplary embodiment, the shell 512 is sufficiently rigid so that when the compartment 538 is filled with liquid (such as water or cell medium), the shell will not bend, flex and / or expand. The shell 512 is usually made of plastics such as polycarbonate, polyolefin, polyester, polystyrene and polyacrylic acid, and can be made of biocompatible materials and can be produced by a molding process such as injection molding, extrusion, blow molding, 3D printing (additive manufacturing), rotational molding or any combination thereof. Forming the shell 512 by plastic also makes the shell relatively cheap, so that it can be discarded or recycled after a single use. The rigid nature of the shell 512 provides stability for the bioreactor 510, and enables it to be self-supported during its different operating modes for correct operation, as discussed below. However, in alternative embodiments, the shell 512 can be formed by a material that has a certain degree of bending, flexing and / or expansion during use but still has sufficient rigidity to self-support. In other alternative embodiments, as further discussed below, the housing 512 may include a collapsible bag made from one or more polymer film sheets supported within a self-supporting, reusable support housing, such as may be made from the same material and have the same properties as the housing 512 discussed above.

[0152] Shell 512 can be elongated and have a height longer than shell width. Specifically, shell 512 has width (e.g., W) or diameter and a height (e.g., H) extending between top end wall 514 and bottom end wall 516. Width can extend between sidewalls 524 and 526, i.e. the width of anterior wall 520, or extends between anterior wall 520 and rear wall 522. Height and width can vary significantly according to the selected volume for compartment 538. In some common embodiments, height is at least or less than 0.2 meter, 0.3 meter, 0.4 meter, 0.6 meter, 0.8 meter, 1 meter or the scope between any two of the foregoing. In some exemplary embodiments, maximum or minimum width or diameter is generally at least or greater than 2.5cm, 5cm, 7.5cm, 10cm, 15cm, 20cm, 30cm, 40cm, 50cm or the scope between any two of the foregoing. In one embodiment, the maximum height H is at least 1.2 times, 1.4 times, 1.6 times, 1.8 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, or in a range between any two of the foregoing values, of the maximum width or diameter. Likewise, other dimensions may be used depending on the intended application. As discussed further below, this elongated configuration may also help optimize mixing efficiency and spraying efficiency when the bioreactor 510 is operated in a dynamic mode.

[0153] In an exemplary embodiment, the housing 512 has approximately the following dimensions: 3.0 inches wide, 3.25 inches deep, and 13.7 inches high, and the compartment 538 has a volume of approximately 1 liter. In another exemplary embodiment, the housing 512 has approximately the following dimensions: 4.8 inches wide, 5 inches deep, and 21.25 inches high, and the compartment 538 has a volume of approximately 5 liters.

[0154] like Fig.21 , the top wall 514 is a cap that can be formed as an integral piece with the side wall 518 or formed separately. The top wall 514 can be fixedly or removably mounted at the upper end of the bioreactor 510 housing 512. The top wall 514 has several features, including: a centered or offset threaded bearing port 530 that can receive the top end 540 of the impeller assembly 550; and a variety of top ports 560 that facilitate aseptic connection with the sensor 580A, dip tube and / or sensor assembly 580B, tubing 580C, fluid transfer system 580A-580C, protective sensor sheath 580A-580C, top sparger or gas overlap assembly (e.g., gas overlap assembly 733 shown in FIG. 25) or other component ports 560. The sensor sheath can be a stainless steel rod, plastic, polymer, rigid or flexible, and can be used to protect the sensor or isolate the sensor from the contents of the bioreactor 510, but also allow the sensor to communicate with the contents of the bioreactor 510 through the opening. The top wall 514 is sealingly engaged with the upper end of the bioreactor 510 housing 512. For example, the top wall 514 can be sealed and / or connected to the top end of the bioreactor 510 housing 512 by welding, adhesives, press-fit connections, use of sealing rings, or other conventional techniques for sealing two parts together and mechanically docking. Spaced-apart optional tubular supports 515 protrude upwardly from the outer surface of the top wall 514, and these spaced-apart optional tubular supports are each configured to receive the hanger 112 ( Fig.11 ), as discussed previously.

[0155] Fig.21The bioreactor 510 depicted in FIG also includes a bioreactor base 590 that is formed as an integral piece with or separately from the sidewall 518. The base 590 can be fixedly or removably mounted at the lower end of the bioreactor 510 housing 512. The base 590 includes several features, including: a sparger port 592 and a sparger 594 for facilitating gas transfer into the compartment 538; a variety of bottom ports 596, including a tube port 596, a discharge port 596, and / or a sampling port 596 for facilitating fluid transfer; a sensor 580D (e.g., a pressure sensor, a temperature sensor, a foam sensor, a glucose sensor, a pH sensor, a DO sensor, a CO2 sensor, a density sensor, a cell density sensor, a conductivity sensor, a point sensor, etc.) for measuring fluid and process parameters; and a leg 599 (e.g., a cylindrical leg) that provides clearance at the bottom of the base 590. In an exemplary embodiment, sensor 580D is a point sensor positioned or affixed to a side wall of bioreactor base 590. Point sensor 580D may also be positioned or affixed to a bottom wall of bioreactor base 590. Sparger port 592 and sparger 594 may be positioned and centered at the surface of base 590, or offset from the center at the surface of base 590 or offset from below impeller assembly 550. Examples of various types of dome sprinklers and film sprinklers that can be used in the present disclosure are disclosed in U.S. Patent Nos. 8,603,805, 9,005,971, 9,259,692, 9,475,012, 9,682,353, 10,328,404, 9,643,133, 10,350,554, 10,843,141, and U.S. Patent Publication No. 2021 / 0069654, which are incorporated herein in their entirety by specific reference. Other conventional sprinklers may also be used. The base 590 is sealingly engaged with the lower end of the bioreactor 510 housing 512. For example, the base 590 can be sealed and connected to the lower end of the bioreactor 510 housing 512 by welding, adhesives, press-fit connections, use of sealing rings, or other techniques for sealing the two parts together and mechanically docking.

[0156] Fig. 22 Depicted Fig.21564 and 565. The top wall or top cap 514 is generally provided with an exterior surface 564 and an opposing interior surface 565. The top wall 514 is mounted at the upper end of the surrounding side wall 518 so that the interior surface 565 faces the compartment 538. More specifically, in an exemplary embodiment, the top wall 514 includes a top panel 566 having an exterior surface 564 and an opposing interior surface 565. A mounting lip 574 projects downwardly from the interior surface 565 so as to be slightly inset from the peripheral edge 572 and form a continuous sub-perimeter. During assembly, the mounting lip 574 is slid into the opening formed at the upper end of the surrounding side wall 518 while the interior surface 565 of the top panel 566 is located on the upper edge of the surrounding side wall 518 of the bioreactor 510 housing 512 ( Fig.21 The mounting lip 574 is sealed to the interior surface of the surrounding side wall 518 ( Fig.21 518) to close the opening of the bioreactor 510 housing 512. The sealing joint can be achieved by welding, adhesives, press-fit connections, use of sealing rings or other techniques. The above-mentioned configuration of the top end wall 514 simplifies the production of both the top end wall 514 and the surrounding side wall 518, and provides easy connection between them. However, it should be understood that the top end wall 514 can also have a variety of other configurations for mounting at the upper end of the surrounding side wall 518.

[0157] The top cap / top end wall 514 of the bioreactor 510 has several features, including: a central or offset threaded bearing port 530 that can receive the top end 540 of the impeller assembly 550 (e.g., the impeller mounting hub 540); and a variety of top ports 560A-560C that facilitate aseptic connection to sensors, dip tubes, tubing, fluid transfer systems, top sparger or gas overlap assemblies, protective sensor sheaths, and / or sampling or component ports. The top ports 560A-560C are connected to the chamber 538 ( Fig.21 For example, sensor 580A and / or sensor sheath ( Fig.21 560A; a dip tube and foam sensor combination 560B can be coupled to and / or housed within port 560B; and a conduit 580C can be connected to port 560C to deliver fluids and / or components to or receive fluids and / or components from compartment 538. Spaced optional tubular brackets 515 project upwardly from the exterior surface of top end wall 514, each of which is configured to receive hanger 112 (see Fig.11 ), as discussed previously.

[0158] In various embodiments, any combination of components 580A-580C and ports 560A-560C may be coupled or integrated. During operation, any of ports 560A-560C may remain closed, open, or blocked during operation of bioreactor 510. Any number and type of sensors 580A may be used during operation and integrated with ports 560A-560C, including temperature sensors, pH sensors, DO (dissolved oxygen) sensors, CO2 sensors, glucose sensors, conductivity sensors, foam sensors, flow sensors, optical sensors, acoustic sensors, electromagnetic sensors, radar sensors, or other sensors capable of detecting bioprocess parameters. Sensor 580A may have a variety of different configurations and may be placed in a variety of different locations on bioreactor housing 512, making top cap 514 customizable under a variety of components and processing conditions.

[0159] One or more spaced apart tube clips 556A-556D are mounted on the top end wall 514 and protrude beyond the top cap 514 and the top panel 566. Each tube clip 556A-556D includes a slot 562, a C-shaped slot 562, a J-shaped slot 562, or a hook 562 configured to removably receive and retain a tube, such as Fig.11 Tubes 234 and 236 are shown. Thus, the tube clips can be used to manage, organize, support and / or straighten tubes used with the bioreactor 510. The tube clips 556A-556D can also be placed in other locations and have other shapes, including open rectangles, open ovals, and similar surrounding structures and shapes.

[0160] Fig.21 The bioreactor 510 and other bioreactors disclosed herein can be operated in a dynamic mode, wherein in the vertical orientation of the bioreactor 510 (e.g. Fig.21 At least some level of mixing occurs in both the vertical (vertical) and horizontal (sideways) orientations shown, and is capable of operating in a static mode where no mixing occurs in both the vertical and horizontal orientations of the bioreactor 510. The bioreactor can effectively grow cells and other biological components in either a vertical or horizontal orientation with periods of mixing, no mixing, or both.

[0161] Fig.23A An example embodiment of an impeller assembly 550 is depicted that can be used to mix the contents of a bioreactor disclosed herein (e.g., bioreactor 10, 510, 710, 1010). The impeller assembly 550 is movably and rotatably disposed within the compartment 538 of the bioreactor 510 ( Fig.21 ), for mechanically mixing the biological components and other fluids in compartment 538 when bioreactor 510 is operated in a dynamic mode. Specifically, as Fig.23AAs shown, the drive shaft 616 is arranged to extend between the top mounting portion 629 and the opposite second end 620. During operation, the drive shaft 616 of the impeller assembly protrudes from the top end wall 514 into the compartment 538 and toward the bottom end wall 516 ( Fig.21 ). The drive shaft 616 may include a central shaft 617 and one or more auxiliary shafts or supports 619 in the form of rods or other structures laterally spaced from the central shaft 617. One or more mixing blades 621 (e.g., three sets of two-blade blades) may be coupled to the central shaft 617 and at least one support structure 619 of the drive shaft 616. In the depicted embodiment, three sets of two-blade blades 621 are connected and coupled to the central shaft 617 and two support rods 619 to form a multi-blade and multi-shaft mixing structure for improved mixing, uniform mixing, and impeller stability.

[0162] In an exemplary embodiment, the impeller assembly 550 further includes an impeller mounting hub 540, which can be used to dynamically couple, mount, and seal the impeller assembly to any bioreactor disclosed herein. For example, the impeller mounting hub 540 can be mechanically coupled or screwed into the bioreactor 510 ( Fig.21 , Fig. 22 510 (or other ports disclosed herein) to partially penetrate the threaded bearing port 530 at the top end wall 514 of the bioreactor 510. A mounting shaft 630 having a terminal end 624 and a bottom mounting portion 627 end extends and protrudes through the impeller mounting hub 540. The bottom mounting portion 627 end of the impeller mounting hub 540 is coupled to the drive shaft 616 of the impeller assembly 550 via a press fit, a hexagonal lock, or other mechanical connection. In an example embodiment, the bottom mounting portion 627 of the impeller mounting hub 540 may include a rod or other component that can be coupled and attached to the top mounting portion 629 of the drive shaft 616.

[0163] The dust cover 622A is located near or adjacent to the terminal end 624 of the impeller mounting hub 540 to accommodate the bearings 636A-636B ( Fig. 23B ) and prevent dust or particles from entering the hub cavity 631 ( Fig. 23B). One or more dynamic seals 622B (e.g., rotatable seals) are located near or adjacent to the bottom mounting portion 627 of the impeller mounting hub 540. In an exemplary embodiment, the dynamic seals 622B include two rotatable lip seals. The dynamic seals 622B seal to form a fluid-tight seal (e.g., an airtight seal) at the bottom of the impeller mounting hub 540 and to create a fluid-tight seal around the hub cavity 631 (shown in 23B). The mounting hub 540 may include a threaded portion 625 to engage and couple to the threaded bearing port 530. The dynamic seal 622B may rotate and enable the drive shaft 616 to rotate within the bioreactor 510 ( Fig.21 530) while sealing the impeller mounting hub 540 and associated ports and preventing external contaminants from entering the mounting hub 540, ports 530 or compartment 538. The terminal end 624 of the impeller mounting hub 540 protrudes into the dust cover 622A and the top end wall 514 ( Fig.21 The impeller mounting hub 540 (and more specifically, the terminal 624) of the drive shaft 116 is connected to the drive motor (e.g., Figure 5 A drive motor 126 is shown coupled, which is typically an electric motor. Figure 5 The drive motor 126 or similar drive motor can be removably mounted on the housing 512 ( Fig.21 ), and more particularly, such as by surrounding the impeller with a hub 540 (in Figure 5 Optional tubular protective sleeve 128 (also depicted as reference numeral 118) Figure 5 ) is mounted to the top wall 514. During use, the drive motor 126 ( Figure 5 ) can be removably coupled to the terminal end 624 of the impeller mounting hub 540 so that activation of the drive motor 126 promotes rotation of the mounting shaft 630 and, in turn, promotes rotation of the drive shaft 616. When the drive shaft 616 is not in use, the drive motor 126 and / or the protective sleeve 128 ( Figure 5 ) can be removed from the housing 512 to minimize the size of the bioreactor 510. An O-ring 633 or other seal can also be incorporated into or positioned on a static portion of the impeller mounting hub 540 (e.g., the stator), such as near the bottom of the impeller mounting hub 540, to prevent rotation of the stator and further seal the compartment 538 and the port 530 to which the impeller mounting hub 540 is coupled.

[0164] Fig. 23BA cross-sectional view of an impeller mounting hub 540 according to an example embodiment is depicted. The impeller mounting hub 540 includes a mounting shaft 630 that extends and protrudes through the impeller mounting hub 540. The mounting shaft 630 has a terminal end 624 and a bottom mounting portion 627 that can be mounted to a drive shaft of an impeller (as described herein) in a compartment of a bioreactor disclosed herein (e.g., 10, 510, 710, 1010). The bottom mounting portion 627 of the impeller mounting hub 540 is coupled to a top mounting portion 629 of a drive shaft 616 or other shaft (e.g., drive shaft 616, 716, 1018) of an impeller assembly disclosed herein (e.g., impeller assembly 550, 750, 1016) via a press fit or other mechanical connection. In an example embodiment, the bottom mounting portion 627 of the impeller mounting hub 540 may include a rod or other component having a specific shape that can be coupled, press-fitted, and attached to the top mounting portion 629 of the drive shaft 616 or any drive shaft disclosed herein (e.g., drive shaft / impeller shaft 616, 716, 1018) in a similar manner. A portion of the mounting shaft 630 engages with one or more bearing assemblies 636A, 636B to facilitate rotation of the mounting shaft 630. A portion of the mounting shaft 630 and the one or more bearing assemblies 636A, 636B are received and sealed within the hub cavity 631 of the mounting hub 540.

[0165] A dynamic seal 622B (e.g., a rotatable seal) is located near or adjacent to the bottom mounting portion 627 of the impeller mounting hub 540. The dynamic seal 622B forms a fluid-tight seal (e.g., an airtight seal) at the bottom of the impeller mounting hub 540 and forms at least a portion of a fluid-tight seal around the hub cavity 631. The mounting hub 540 may include a threaded portion 625 to engage and couple to the threaded bearing port 530 (or threaded bearing port 730 shown in FIG. 25). The dynamic seal 622B may rotate and enable the mounting shaft 630 and one or more bearing assemblies 636A, 636B to rotate within the hub cavity 631 while sealing the impeller mounting hub 540 and associated ports and preventing external contaminants from entering the mounting hub 540, the ports 530, or the compartment 538 ( Fig.21 ). The terminal end 624 of the impeller mounting hub 540 protrudes outside of the dynamic seal 622B. The impeller mounting hub 540 (and more specifically, the terminal end 624) can be coupled to a drive motor, which is typically as described with respect to Figure 5 and Fig.23A During use, the impeller mounting hub 540 and the mounting shaft 630 may be coupled to the drive motor 126 ( Figure 5540) to rotate the drive shaft 127 of the impeller assembly 550 to rotate the mounting shaft 630 and, in turn, the drive shaft 616 of the impeller assembly 550. An O-ring 633 or other seal may also be incorporated into or positioned on a static portion of the impeller mounting hub 540 (e.g., stator), such as near the bottom of the mounting hub 540, to prevent movement / rotation of the actuator and to further seal the compartment 538 and the port through which the mounting hub 540 is coupled. The impeller mounting hub 540 can be used interchangeably and can be used in conjunction with any of the mixing elements, mixers, impeller assemblies, drive shafts, and bioreactors disclosed in any of the embodiments depicted in the figures.

[0166] FIG. 24A to FIG. 24B A top view and a perspective view of a bioreactor base 590 according to an exemplary embodiment are depicted, respectively. The bioreactors disclosed herein and depicted in the accompanying drawings may include a variety of bioreactor bases, such as FIG. 24A to FIG. 24B The bioreactor base 590 depicted in FIG. The bioreactor base 590 may be attached to the sidewalls of the bioreactor (e.g., Fig.21 The base 590 may be fixedly or removably mounted on a bioreactor housing (e.g., Fig.21 The base 590 has side walls 507 and a bottom wall 509. The base 590 includes several features, including a sparger port 592 and a sparger 594 for facilitating gas transfer to the bioreactor compartment (e.g., Fig.21 590 ); a plurality of bottom ports 596, including a tube port 596 for facilitating fluid transfer, an exhaust port 596, a sparger gas port 596, and / or a sampling port 596; a sensor 580D (e.g., a pH sensor, a DO sensor, a conductivity sensor, a pressure sensor, a temperature sensor, a foam sensor, a point sensor, etc.) for measuring fluid and process parameters; and a leg 599 (e.g., a cylindrical leg) that provides clearance at the bottom of the base 590. In an example embodiment, the sensor 580D can be a point sensor coupled to the side wall 507 or the bottom wall 509 of the bioreactor base 590. The sensor 580D can also be other sensors or probes coupled to ports in the base 590. The sparger port 592 and the sparger 594 can be positioned at the surface of the base 590 and centered or offset at the surface of the base 590. In an example embodiment, the sparger port 592 and the sparger 594 are located at the impeller assembly (e.g., Fig.21 Any of the bioreactors disclosed herein may be docked with, attached to, or assembled into a bioreactor base 590. For example, the base 590 may be attached to a bioreactor housing (e.g., Fig.21The base 590 may be sealed and connected to the bioreactor housing (e.g., housing 512) by welding, adhesives, press fit connections, use of sealing rings, or other techniques for sealing and mechanically docking two parts together. Fig.21 The lower end of the bioreactor 510 and the housing 512).

[0167] FIG. 25A to FIG. 25B A perspective view of a bioreactor 710 and a top view of a bioreactor base 790 are depicted, respectively, according to an exemplary embodiment. The bioreactor 710 has a number of Fig.21 , including a housing 712 having a top end wall 714, an opposing bottom end wall 719, and a surrounding side wall 718 extending therebetween. In the depicted embodiment, the surrounding side wall 718 has a rectangular or square transverse cross-section. However, in other embodiments, the housing 712 and the surrounding side wall 718 may have other transverse cross-sectional configurations, such as circular, elliptical, polygonal. In an exemplary embodiment, the housing 712 has a cubic shape. In other exemplary embodiments, the housing 712 and the top end wall 714 have a foolproof feature, wherein one corner has a different radius of curvature than the other corners as described herein.

[0168] Shell 712 has the inner surface that defines compartment 738.The volume of compartment 738 can be at least or less than 50 milliliters, 100 milliliters, 250 milliliters, 500 milliliters, 1 liter, 5 liters, 10 liters, 20 liters, 30 liters, 40 liters, 50 liters or in the scope between any two of the foregoing.For example, compartment 738 has the volume in the scope between 250 milliliters and 50 liters conventionally, wherein between 1 liter and 20 liters or between 1 liter and 10 liters is more common.Also other volumes can be used.In one embodiment, shell 712 and wall thereof are made of gas and liquid (such as medium) impermeable material.In other examples, compartment 738 can have the volume greater than 50 liters.

[0169] In addition, shell 712 and its wall are rigid in general.For example, in an exemplary embodiment, shell 712 is sufficiently rigid so that when compartment 738 is full of liquid (such as water or medium), shell will not bend, flex and / or expand.Shell 712 is usually made of plastics such as polycarbonate, polyolefin, polyester, polystyrene, polyacrylic acid, biocompatible material and / or transparent material, and can be produced by a molding process such as injection molding, extrusion, blow molding, 3D printing (additive manufacturing), rotational molding or any combination thereof.Forming shell 712 by plastic also makes shell relatively cheap so that it can be discarded or recycled after single use.The rigidity of shell 712 provides stability for bioreactor 710, and enables it to be self-supported during its different operating modes to carry out correct operation, as discussed below.However, in alternative embodiments, shell 712 can be formed by a material that has a certain degree of bending, flexing and / or expansion during use but still has sufficient rigidity to self-support. In other alternative embodiments, as discussed further below, the housing 712 may include a collapsible bag made of one or more sheets of polymer film supported within a self-supporting, reusable support housing, such as may be made of the same materials and have the same properties as the housing 712 discussed above. The housing 712 may be made of a transparent material to allow visual inspection of the contents and internal components of the bioreactor 712.

[0170] In an exemplary embodiment, the housing 712 has approximately the following dimensions: 3.0 inches wide, 3.25 inches deep, and 13.7 inches high, and the compartment 738 has a volume of approximately 1 liter. In another exemplary embodiment, the housing 712 has approximately the following dimensions: 4.8 inches wide, 5 inches deep, and 21.25 inches high, and the compartment 738 has a volume of approximately 5 liters.

[0171] As depicted in FIG. 25 , the top wall 714 is a cover or cap that can be formed as an integral piece with the side wall 718 or formed separately. The top wall 714 can be fixedly or removably mounted at the upper end of the bioreactor 710 housing 712. The top wall 714 has several features, including but not limited to: a centered or offset threaded bearing port 730 that can receive the impeller mounting hub 740 of the impeller assembly 750; and a variety of top ports 760 that facilitate aseptic connection with sensors, sprayers, gas syringes or stacks, dip tubes and / or sensor combinations, tubing, fluid transfer systems, protective sensor sheaths, or other component ports 760. The sensor sheath can be a stainless steel rod, plastic, polymer, rigid or flexible, and can be used to protect or isolate a sensor or sensor component from the contents of the bioreactor 710. In an exemplary embodiment, the sensor assembly 780 is hermetically coupled to the top port 760 and inserted into the compartment 738 through the port 760. In an example embodiment, the sensor assembly 780 is coupled to the PG 13.5 port 760 and includes three foam sensor sticks surrounding the foam sensor (or level sensor) and two dip tubes for other sensors (e.g., thermocouples) to be inserted in the dip tubes or for directing fluid flow into the bioreactor 710. In other example embodiments, the sensor assembly 780 includes three foam sensor sticks capable of surrounding the foam sensor and one dip tube with a resistance temperature detector (RTD) inserted.

[0172] Additionally, the top wall 714 can include a gas injector 733 (such as an overhead sparger 733 or a gas overlap assembly 733) that is fluidly coupled to the sparger or gas injector port 760A. The gas overlap assembly 733 can be connected to a gas source to facilitate the flow of gas out of or into the bioreactor 710. The gas overlap assembly 733 can include a tubular conduit 733A, a gas outlet nozzle 733B, a valve 733C for regulating the flow of gas through the conduit 733A and out of the nozzle 733B, and a filter 733D for filtering any incoming gas or particles and contaminants. For example, the gas overlap assembly 733 can allow oxygen (e.g., oxygen cross-flow) to flow to the head space of the bioreactor 710 and flow across the top liquid surface within the bioreactor 710 to supply and oxygenate the biological components within the biological fluid (e.g., cell culture) in the bioreactor 710. The concentration of oxygen flowing to the head space of the bioreactor 710 can also control the pH of the biological fluid. The gas overlap assembly 733 can also remove carbon dioxide from the headspace of the bioreactor 710 by connecting the gas overlap assembly 733 to a pump to pump out gas. The gas overlap assembly 733 can ensure that oxygen reaches the biological fluid within the bioreactor 710 when the bioreactor 710 is operated in a static mode and at a low turndown ratio when the bioreactor is not full of biological fluid (such as a cell culture). The gas overlap assembly 733 can supply oxygen to the bioreactor and cell culture when the cell culture is 5% to 90% full relative to the volume of the bioreactor as measured by the volume of the cell culture. The overhead sparger, gas overlap assembly, its components and functionality are referred to as a gas delivery system and are described in detail in U.S. Patent Nos. 9,388,375, 9,932,553, 10,519,413, 11,162,062, which are incorporated herein by reference in their entirety. The gas delivery systems and gas overlap assemblies 733 described herein and in US Pat. Nos. 9,388,375, 9,932,553, 10,519,413, 11,162,062 may be coupled to the top port 760A of any of the bioreactors described herein.

[0173] The top end wall 714 is sealingly engaged with the upper end of the bioreactor 710 housing 712. For example, the top end wall 714 can be sealed and / or connected to the top end of the bioreactor 710 housing 712 by welding, adhesives, press fit connections, use of sealing rings, or other techniques for sealing and mechanically abutting two parts together.

[0174] FIG. 25A to FIG. 25B The bioreactor 710 depicted in FIG. 7 also includes a bioreactor base 790 ( Fig.25B), the bioreactor base is formed as an integral piece or separately from the sidewall 718. The base 790 can be fixedly or removably mounted at the lower end of the bioreactor 710 housing 712. The base 790 includes several features, including: a sparger port 792 and a sparger 794 for facilitating gas transfer into the compartment 738; a variety of bottom ports 796, including a tube port 796, a discharge port 796, and / or a sampling port 796 for facilitating fluid transfer; a sensor 780A (e.g., a pH sensor, a DO sensor, a conductivity sensor, an infrared temperature sensor, a foam sensor, or a point sensor) for measuring fluid and process parameters; and a leg 799. The leg 799 can be a flat, square, or rectangular leg that provides a gap at the bottom of the base 790. Each leg 799 can span the entire length of the side wall 718 of the bioreactor 710 (e.g., as shown in FIG. 25 ) to provide additional support and facilitate placement and coupling of the legs 799 to a heater support (e.g., Figure 11 to Figure 12 heater support 202) or facilitates the connection and insertion of the bioreactor 710 into the FIG. 29A to FIG. 29B The bioreactor pod base 1002 is depicted. The legs 799 of the bioreactor base 790 can also be positioned on surfaces such as tables and on top of and inside other equipment such as incubators.

[0175] The sparger port 792 and sparger 794 are offset from the center of the bottom wall 709 of the base 790. The sparger 794 is offset from the center of the base 790 and from the center of the impeller assembly 750 to allow more bubble entrainment and prevent possible impeller cavitation. The base 790 is sealed and engaged with the lower end of the bioreactor 710 housing 712. For example, the base 790 can be sealed and connected to the lower end of the bioreactor 710 housing 712 by welding, adhesives, press-fit connections, using sealing rings or other techniques for sealing two parts together and mechanically docking. The bioreactor base 790 includes a wall 711 that is raised or angled relative to the bottom wall 709 of the base 790. The sensor 780A can be positioned or coupled to the angled wall 711 to prevent cell accumulation near or close to the sensor 780A. Cell accumulation around the sensor 780A can cause inaccurate readings and defective sensor measurements, thereby causing incorrect fluid properties and process parameter outputs associated with the bioreactor 710.

[0176] In example embodiments, when the bioreactor 710 is in a vertical orientation, the angled wall 711 is at an angle between 5 and 70 degrees relative to the planar surface 713 of the bioreactor base 790 or relative to a horizontal plane across the bioreactor 710. In other example embodiments, when the bioreactor 710 is in a vertical orientation, the angled wall 711 is at an angle of 45 degrees relative to the planar surface 713 of the bioreactor base 790 or relative to a horizontal plane across the bioreactor 710.

[0177] In an exemplary embodiment, the drain port 796 is oriented vertically to facilitate gravity-assisted draining of liquid. In an exemplary embodiment, the drain port 796 is 4 mm to 12 mm in length to prevent liquid retention in the compartment 738 of the bioreactor 710 or to reduce liquid holdup volume.

[0178] Fig.25A The bioreactor 710 and other bioreactors disclosed herein are capable of operating in a dynamic mode, wherein at least some level of mixing occurs in both the vertical and horizontal orientations of the bioreactor 710, and are capable of operating in a static mode, wherein no mixing occurs in both the vertical and horizontal orientations of the bioreactor 710. The bioreactors can effectively grow cells and other biological components disposed vertically or horizontally with mixing, no mixing, or alternating periods of mixing and no mixing.

[0179] The impeller assembly 750 of the bioreactor 710 can be used to mix the contents of the bioreactor 710 and other bioreactors disclosed herein. The impeller assembly 750 is movably disposed within the compartment 738 of the bioreactor 710 for mechanically mixing the biological components and other fluids within the compartment 738 when the bioreactor 710 is in a dynamic mode. Specifically, as Fig.25A As shown, the impeller assembly 750 includes a drive shaft 716, a mixing blade 721, and an impeller mounting hub 740 (having a FIG. 23A to FIG. 23BThe drive shaft 716 may include three sets of three-leaf blades or two-leaf blades 721. The drive shaft 716 extends between the impeller mounting hub 740 and the opposite second end 720 within the compartment 738. The drive shaft 716 protrudes from the top end wall 714 into the compartment 738 and toward the base 790 of the bioreactor 710. The drive shaft 716 may include one or more mixing blades 721 (e.g., two-leaf blades or three-leaf blades) coupled to the drive shaft 716. The blades may have the same or similar size and configuration as the blades described in U.S. Patent Nos. 9,855,537, 10,335,751, 11,654,408, 9,839,886, 10,272,400 and U.S. Patent Publication Nos. 2019 / 209,981 and 2021 / 237,009, which are incorporated herein by reference in their entirety.

[0180] In an exemplary embodiment, the impeller mounting hub 740 may be used to FIG. 23A to FIG. 23B The impeller assembly 750 of the bioreactor 710 may be dynamically coupled, mounted and sealed in the same manner as described above. For example, the impeller mounting hub 740 may be mechanically coupled or screwed into the bioreactor 710 ( Fig.25A 724) to partially extend through the threaded bearing port 730 at the top end wall 714 of the bioreactor 710. The mounting shaft 731 protrudes through the impeller mounting hub 740. The bottom end of the mounting shaft 731 is coupled to the drive shaft 716 of the impeller assembly 750 via a press fit or other mechanical connection. The impeller mounting hub 740 (more specifically, the terminal end 724) can be connected to a drive motor (e.g., Figure 5 The drive motor 126 shown is connected, as shown in Figure 5 and FIG. 23A to FIG. 23B In an exemplary embodiment, the mounting hub 740 of FIG. 25 has a Fig.21 , FIG. 23A to FIG. 23B The mounting hub 540 has the same features and elements.

[0181] Fig.26800 according to an example embodiment. The sprinkler 800 includes a sprinkler disk 802 having apertures 804 of a particular hole size. The holes / apertures 804 in the sprinkler disk 802 may be laser drilled and may have the following number of holes and sizes: 360×178 μm holes; 570×178 μm holes; 760×233 μm holes; 980×368 μm holes; 1,180×445 μm holes. In an example, the sprinkler disk 802 has a diameter of 20 mm to 60 mm, the sprinkler aperture range is 20 μm to 140 μm, and the sprinkler disk has 1 to 1,000 apertures or holes. In other embodiments, a combination of holes 804 of different sizes are drilled in the sprinkler disk 802. The sparger 800 interfaces with or includes a gas line 806 to supply gas through the sparger disk 802 and into the compartment of the bioreactor. The sparger 800 can be used with any bioreactor disclosed herein to transfer gas to the components of the bioreactor. The sparger can also have a non-circular geometry to better utilize and save space at the base of the bioreactor disclosed herein. The sparger 800 can also have the same features as the sparger 166 or sparger 168 previously described. Examples of various types of dome and film sprinklers that can be used in the present disclosure are disclosed in U.S. Patent Nos. 8,603,805, 9,005,971, 9,259,692, 9,475,012, 9,682,353, 10,328,404, and 9,643,139,643,133, 10,350,554, 10,843,141, and U.S. Patent Publication No. 2021 / 0069654, which are incorporated herein by specific reference. Other conventional sprinklers may also be used.

[0182] Fig. 27Depicted is the bottom portion of a bioreactor base 790 according to an example embodiment. The bioreactor base 790 can be any bioreactor base disclosed herein with a discharge or sampling port, including the bioreactor base 790 depicted in FIG. 25 . The bioreactor base 790 includes a discharge or sampling port 796A and a discharge or sampling tube 796B coupled to the port 796A. Biological components (and specifically, liquid from the interior of the bioreactor) can be discharged through the discharge or sampling port 796A and the discharge or sampling port 796B. A one-way valve 796C can be coupled to the port 796A or tube 796B at the top or bottom of the port 796A or tube 796B. The one-way valve 796C only allows fluid to flow in one direction to prevent fluid from re-entering the port 796A through the one-way valve 796C after being discharged from the port 796A and the base 790 of the bioreactor 710. In an example embodiment, the one-way valve 796C can be a check valve, a ball valve, a duckbill valve, an umbrella valve, or other one-way valve to prevent liquid from re-entering port 796A. The umbrella valve can be modified to allow fluid to flow in only one direction, as disclosed in U.S. Patent No. 11,598,434 issued on March 7, 2023 and U.S. Publication No. US2023184347A1 published on June 15, 2023, which are incorporated herein by specific reference. Fig. 27 The port, tube and valve configuration depicted in reduces dead space and liquid hold-up volume in the base 790 of the bioreactor disclosed herein. Fig. 27 Also depicted are legs 799 attached to the bioreactor base 790. The legs 799 can be flat, square, or rectangular legs that provide clearance at the bottom of the base 790. Each leg 799 can span the entire length of the sidewall 718 of the bioreactor 710 (e.g., Fig.25A ), to provide additional support and facilitate placement, coupling or sliding of the legs 799 to a heater support (e.g., Figure 11 to Figure 12 heater support 202) or bioreactor chamber base 1002 (in Fig.29A as shown in ).

[0183] FIG. 28A to FIG. 28C Depicted are sensor and port sets 900A-900C that enable coupling, protection, and operation of sensors at the top wall or other walls (eg, side walls) of the bioreactors disclosed herein. FIG. 28A to FIG. 28CVarious configurations of dual-mode bioreactors 910A-910C that can operate in static and dynamic modes are depicted in FIG. The dual-mode bioreactors 910A-910C have various impeller assemblies 950A-950C and sensor and port groups 900A-900C used during operation of the bioreactors 910A-910C. Each sensor and port group 900A-900C includes one or more ports, sensor hubs, sensor sheaths, dip tubes, sleeves, and / or sensors therein. For example, Fig.28A The sensor and port set 900A includes a resistance temperature detector (RTD) sheath 980A that is coupled to the port 960A at the top wall or cap of the bioreactor 910A (e.g., via a threaded connection). A resistance temperature detector (e.g., a thermocouple or a temperature probe) is inserted into the sensor sheath 980A to isolate the sensor / detector from the contents of the bioreactor 910A while a temperature measurement is generated by the sensor or its probe from an opening in the sensor sheath 980A.

[0184] Fig.28B The sensor and port group 900B includes a sensor hub 911B, which is connected to the sensor port 960B at the top wall or cap of the bioreactor 910B (e.g., via a threaded connection). The sensor hub 900B includes at least two holes. The dip tube 980B and the foam sensor 981B in the dip tube 980B are inserted into each hole. The foam sensor 981B (e.g., a liquid level sensor) can be connected to the contents of the bioreactor 910B through the opening at the bottom of the dip tube 980B to sense the accumulation, volume and / or height of the foam in the bioreactor 910B. The dip tube 980B may include a sensor or provide a fluid path to allow the fluid / liquid to flow and be discharged into a specific discharge position in the bioreactor 910B.

[0185] Fig.28CThe sensor and port group 900C includes a port 960C and a dissolved oxygen sleeve 980C, which is connected to the port 960C at the top wall or cap of the bioreactor 910C (e.g., via a threaded connection). The dissolved oxygen sensor is inserted into the sleeve 980C to isolate the sensor from the contents of the bioreactor 910C, while the dissolved oxygen measurement is generated by the sensor near the bottom of the sleeve 980C (e.g., through an opening in the sleeve 980C). The sensor sheath 980A, sleeve 980C, and dip tube 980B can be made of rigid or flexible materials, including rigid or flexible tubes or hollow rods made of steel, stainless steel, polymeric materials or other metals, plastics, or medical grade / surgical / sterile materials. The sensor sheath 980A, sleeve 980C, and dip tube 980B may also include an opening in the sheath / sleeve / tube to facilitate communication between the sensor and the contents of the dual-mode bioreactor 910A-910C.

[0186] FIG. 28A to FIG. 28C The dual-mode bioreactors 910A-910C and other bioreactors disclosed herein are capable of operating in a dynamic mode, wherein at least some level of mixing occurs in both the vertical and horizontal orientations of the bioreactors 910A-910C, and are capable of operating in a static mode, wherein no mixing occurs in both the vertical and horizontal orientations of the bioreactors 910A-910C. The bioreactors 910A-910C can effectively grow cells and other biological components disposed vertically or horizontally during periods of mixing, no mixing, or both mixing and no mixing to customize mixing, aeration, sparging, and other operations for cell therapy, gene therapy, antibody production, or other applications of growing and producing biological components.

[0187] FIG. 29A to FIG. 29BA front view and a rear view of a bioreactor pod 1000 according to an example embodiment are depicted, respectively. The reactor pod 1000 may include: a pod base 1002; a dual-mode bioreactor 1010 (e.g., any of the bioreactors described herein) that may be inserted into the pod base 1002; one or more portable and deployable pod modules 1004, 1006, 1008, 1017; a bracket 1012 for mounting pod components; a display 1014 that includes a user interface for sending user input and receiving input from the bioreactor; and a display 1015 that includes a user interface for sending user input and receiving input from the bioreactor. The controller 1037 includes a bioreactor pod 1000, a bioreactor 1010, pod modules 1004, 1006, 1008, 1017, and pod components and process parameter outputs associated therewith; a motor 1024; a sensor 1080; and a controller 1037 having a memory and a processor, located in the pod base 1002 and used to control the bioreactor 1010, the motor 1024, the pod modules 1004, 1006, 1008, 1017, and other pod 1000 components. In other embodiments, the controller 1037 can be a separate unit from the pod base 1002 and located away from the bioreactor pod 1000.

[0188] User inputs associated with the bioreactor 1010 or bioreactor pod 1000 may include, but are not limited to, set points or user inputs indicating: fluid temperature, system pressure, impeller speed, motor RPM, gas flow rate into or out of the bioreactor, dissolved oxygen concentration in a liquid (e.g., cell culture medium), pH of the liquid, liquid flow rate into or out of the bioreactor; gas flow rate through a bottom sparger; gas flow rate through a gas overlap assembly; pump speed or RPM, pump direction, a barcode scanned by a scanner 1031, and / or an operating pod / bioreactor recipe that controls the duration of static and dynamic growth times / conditions in the bioreactor and controls bioreactor pod components such as pod modules 1004, 1006, 1008, 1017. All or some of these inputs may be stored in a workflow or pod recipe that is selected by operator input at the display 1014 and user interface and executed by the controller 1037 processor, as described herein.

[0189] Process parameter outputs associated with the bioreactor 1010 and bioreactor pod 1000 may include, but are not limited to, sensor measurements, data, or signals; impeller speed; motor RPM; gas flow rate into or out of the bioreactor, liquid flow rate into or out of the bioreactor; gas flow rate through the bottom sparger; gas flow rate through the gas overlap assembly; pump speed or RPM; and / or scanner data (e.g., scanner 1031 described below). A variety of sensors (e.g., sensor 1080) may be coupled to the bioreactor 1010, including, but not limited to, pressure sensors, temperature sensors, foam sensors, glucose sensors, pH sensors, DO sensors, CO2 sensors, flow sensors, density sensors, cell density sensors, conductivity sensors, point sensors, and the like. The data or signals measured by the sensor and sent from the sensor (or associated transmitter) are fluid and system parameter outputs such as pressure within a bioreactor, temperature within a bioreactor or cell culture; foam content within a bioreactor; glucose content within a bioreactor or cell culture; pH within a bioreactor or cell culture; dissolved oxygen content within a bioreactor or cell culture; CO2 content within a bioreactor or cell culture; cell density of a cell culture; conductivity readings associated with a bioreactor or cell culture.

[0190] The bioreactor 1010 is a dual mode bioreactor 1010 that can operate in a static mode (no mixing occurs) and a dynamic mode (at least some mixing or agitation occurs) to support a biologically active environment and perform biological processes, such as seed culture and cell expansion applications. The time periods and alternating durations of the static mode operation and dynamic mode operation of the bioreactor 101 can be stored as a recipe in the memory of the controller 1037 of the bioreactor pod 1000 to optimize cell growth and preservation.

[0191] In an example embodiment, the bioreactor 1010 may include the same features and components as the bioreactor 710 depicted in FIG25. In this example of the bioreactor pod 1000, the bioreactor 1010 includes an impeller assembly 1016 having one impeller shaft 1018 (or drive shaft) and three groups of three impeller blades 1020 (or three-blade blades) vertically spaced and coupled to three segments of the impeller shaft 1018, resulting in a total of nine blades coupled to the impeller shaft 1018. In an example embodiment, one to six groups of three-blade blades 1020 are vertically spaced and coupled to one or more segments of the shaft 1018. The impeller assembly 1016 may also include an impeller mounting hub 1022 ( Fig.29B) to secure and couple one end of the impeller shaft 1018 to the motor 1024. In an example embodiment, the impeller shaft 1018 may be coupled to the electric motor 1024 and / or an associated motor shaft via the impeller mounting hub 1022. During operation of the bioreactor 1010 in a dynamic mode, the motor 1024 rotates the shaft of the impeller mounting hub 1022, which in turn rotates the impeller shaft 1018 and the three-blade blade 1020 of the impeller assembly 1016. The motor mount 1035 is attached to the bracket 1012. When the motor mount 1035 is detached from the impeller mounting hub 1022, the motor mount can be used to mount the motor 1024. The impeller mounting hub 1022 can be any impeller mounting hub described herein (e.g., FIG. 23A to FIG. 23B 25). In an exemplary embodiment, the impeller mounting hub 1022 has the same FIG. 34A to FIG. 34C The impeller mounting hub 1500 has the same components, structure and functionality as depicted in FIG.

[0192] The top end wall 1026 of the bioreactor 1010 is a cover or cap that can be formed as an integral piece with or separately from the housing 1028 of the bioreactor 1010, as described with respect to FIG25. The top end wall 1026 can be fixedly or removably mounted at the upper end of the bioreactor housing 1028. The top end wall 1026 has several features, including, but not limited to: a centered or offset threaded bearing port 1030 that can receive the impeller mounting hub 1022 of the impeller assembly 1016; and a variety of top ports 1060A, 1060C that facilitate aseptic connection to sensors, sparger, gas overlap assembly, dip tube and / or sensor combination, tubing, fluid transfer system, protective sensor sheath or other component ports 1060A, 1060C. The sensor sheath can be a stainless steel rod, plastic, polymer, rigid or flexible, and can be used to protect the sensor or isolate the sensor from the contents of the bioreactor 1010, and also provide an opening for the sensor to communicate with the contents of the bioreactor 1010. In an exemplary embodiment, the sensor assembly 1080 is hermetically connected to the top port 1060A and inserted into the bioreactor compartment 1038 through the port 1060A. In an exemplary embodiment, the sensor assembly 1080 (or sensor group) is connected to the PG 13.5 port 1060A and includes three foam sensor rods surrounding the foam sensor and two dip tubes. In other exemplary embodiments, the sensor assembly 1080 includes three foam sensor rods capable of surrounding the foam sensor, a dip tube, an RTD tube, and a barb and cable tie connection that connects the sensor group to the cap 1026 of the bioreactor 1010. In an exemplary embodiment, the sensor assembly (or sensor group) is about FIG. 31A to FIG. 31D Sensor assembly 1080 is described.

[0193] Overhead sprinkler or gas overlap assembly 1033 ( Fig.29A ) can be fluidly coupled to gas port 1060C ( Fig.29B 25 ). The gas overlap assembly 1033 can be connected to a gas source via the gas flow module 1008 to facilitate gas flow out of or into the bioreactor 1010. The gas overlap assembly 1033 can include a tubular conduit, a gas outlet nozzle, a valve, and a filter (as shown in FIG. 25 ) to regulate the flow of gas passing through the conduit and out of the nozzle. For example, the gas overlap assembly 1033 can allow oxygen (e.g., oxygen cross-flow) to flow to the head space of the bioreactor 1010 and flow through the top liquid surface within the bioreactor 1010 to supply and oxygenate the biological components in the biological fluid (e.g., cell culture) in the bioreactor 1010. The concentration of oxygen flowing to the head space of the bioreactor 1010 can also control the pH of the biological fluid. The gas overlap assembly 1033 can also remove carbon dioxide from the head space of the bioreactor 1010 via the pod module 1008 or by connecting the gas overlap assembly 1033 to a pump to pump out the gas. The gas overlap assembly 1033 can ensure that oxygen reaches the biological fluid within the bioreactor 1010 when the bioreactor 1010 is operated in static mode or dynamic mode and when the bioreactor is not full of biological fluid (such as cell culture medium) when operating at a low turndown ratio. The gas overlap assembly 1033 can supply oxygen to the bioreactor 1010 and the cell culture when the bioreactor 1010 is 5% to 90% full of cell culture medium (as measured by the volume of the cell culture relative to the volume of the bioreactor 1010). The gas overlap assembly 1033, the overhead sparger, its components and functionality are referred to as a gas delivery system and are described in detail in U.S. Patent Nos. 9,388,375, 9,932,553, 10,519,413, 11,162,062. The gas delivery systems, overhead spargers, and gas overlap assemblies 1033 described herein and in US Pat. Nos. 9,388,375, 9,932,553, 10,519,413, 11,162,062 may be coupled to the top port 1060C of any of the bioreactors described herein.

[0194] The bioreactor 1010 also includes a bioreactor base 1090 having FIG. 25A to FIG. 25B and Fig. 27 The same or similar components, features and functionality as described above may be used with the bioreactor base 790. FIG. 25A to FIG. 25B and Fig. 27 As more clearly shown in FIG. 25 , the bioreactor base 1090 has legs 799 ( FIG. Fig. 27), these legs can slide into and couple to the bioreactor receiver 1092 of the bioreactor pod base 1022 to secure the bioreactor 1010 to the bioreactor pod 1000. The bioreactor base 1090 also has a bottom sparger 1094 to flow gas (e.g., oxygen) to the contents of the bioreactor 1010 when the bioreactor is operated in a dynamic or static mode. The bottom sparger 1094 can be any sparger described herein, including but not limited to: Figure 8 The described sprinklers 166 and / or 168; Fig.13 The described sprinkler 222; Fig.21 , the sprinkler 594 described in FIG. 24; the sprinkler 794 described in FIG. 25; or Fig.26 The sparger 800 described above. Examples of various other sparger types that may be used in the present disclosure and as sparger 1094 are disclosed in U.S. Pat. Nos. 9,005,971 and 9,643,133, which are incorporated herein by specific reference. Other conventional sparger types may also be used. The bottom sparger 1094 may be offset from the center of the bioreactor base 1090, as described with respect to FIG. Fig.21 1016. The sparger 1094 may be centered below the impeller assembly 1016 as described with respect to the sparger 594. Offsetting the sparger 1094 from directly below the impeller assembly 1016 may prevent the collapse and / or coalescence of the sparger bubbles, which may damage or kill cells and inhibit cell growth.

[0195] The pod base 1002 may include a number of Fig.11 , Fig.12 , Fig.16 and Fig.17 The pod base 1002 includes a controller 1037 having a memory and a processor, located in the pod base 1002 and used to control the bioreactor 1010, the motor 1024, the pod modules 1004, 1006, 1008, 1017 and other pod 1000 components. In other embodiments, the controller 1037 can be a separate unit from the pod base 1002 and located away from the bioreactor pod 1000. The pod base 1002 also includes the components described with respect to Fig.11The heating element 291 of the heater support 202 described above is the same as or similar to a heating element (not shown). The heating element 291 forms a heater with the bioreactor receiver 1092 that can heat the contents of the bioreactor 1010. The heating element can be a resistive heating element, a conductive heating element, or other heating element, and the bioreactor receiver 1092 can be made of a conductive material (e.g., aluminum, titanium, or other conductive material) that conducts heat from the heating element to the bioreactor base 1090 and the contents of the bioreactor 1010. In an exemplary embodiment, the heating element is incorporated into the bioreactor receiver 1092. The bioreactor receiver 1092 can be formed as a mold of a portion of the bioreactor 1010 housing 1028 to optimally dock with and hold the bioreactor 1010. The bioreactor receiver 1092 can also have an opening 1093 into which the bioreactor 1010 can be slid to mount the bioreactor 1010 to the pod base 1002. The opening 1093 also provides visibility of the bioreactor 1010 and the contents of the bioreactor 1010 through the transparent housing 1028 of the bioreactor 1010 .

[0196] The pod base 1002 may also include a power supply unit 1040 that can supply power to components of the bioreactor pod 1000 (including, but not limited to, pod modules 1004, 1006, 1008, 1017, motor 1024, and sensor 1080) and controller 1037 via a cable 1041. The cable 1041 can transmit power and / or data to and from components of the bioreactor pod 1000 (including, but not limited to, pod modules 1004, 1006, 1008, 1017, motor 1024, and sensor 1080) and controller 1037. Control, sensor, and component signals and data may also be wirelessly transmitted between components of the bioreactor pod 1000 and controller 1037 to control the operation of the bioreactor pod 1000 and its components. The power supply unit 1040 may include one or more AC power supplies, DC power supplies, distribution boxes, programmable power supplies, uninterruptible power supplies, switch mode power supplies, and / or other power supplies.

[0197] The pod modules 1004, 1006, 1008, 1017 are modular components, each being a separate unit or one or more combined units, which are portable, stackable and can be arranged in a variety of configurations. The pod modules 1004, 1006, 1008, 1017 can be stacked on top of each other in any order. The pod modules 1004, 1006, 1008, 1017 can also be arranged individually on a surface without stacking. The pod modules 1004, 1006, 1008, 1017 can be positioned on the right or left side of the bioreactor 1010 and the pod base 1002 in a stacked manner or individually in a non-stacked manner. One or more of the pod modules 1004, 1006, 1008, 1017 may be positioned on the left side of the bioreactor 1010 and the pod base 1002, while one or more of the pod modules 1004, 1006, 1008, 1017 are positioned on the right side of the bioreactor 1010 and the pod base 1002. The pod modules 1004, 1006, 1008, 1017 include notches 1021, recessed portions 1021, or flanges 1021 to allow a user to easily handle, lift, move, and arrange the pod modules 1004, 1006, 1008, 1017 in any stacked or non-stacked configuration or position. Each of the pod modules 1004, 1006, 1008, 1017 and / or an associated pod transmitter may communicate with, send data to, and receive data and control signals from the controller 1036 to control the operation of the pod modules 1004, 1006, 1008, 1017. The data and signals may be transmitted wirelessly via a wireless transmitter or wired via a cable 1041 between the pod modules 1004, 1006, 1008, 1017 and the controller 1037.

[0198] The bioreactor pod 1000 may include any number of pod modules, including, for example: 1 to 10 pump modules for pumping fluids to / from the pod equipment including the bioreactor; 1 to 10 mass flow controller modules for controlling the flow of fluids to / from the pod equipment; 1 to 10 electrical modules for powering the pod equipment; 1 to 10 equipment control modules for controlling the pod equipment; 1 to 10 defoaming modules for deploying foam control measures; 1 to 10 sensor transmitter modules that receive, process and transmit sensor data, signals and measurements; 1 to 10 emergency stop modules that can control and cut off power to the pod equipment; 1 to 10 heater modules for heating the pod equipment including the bioreactor; and / or other modules that are stacked, arranged and customized for specific expansion processes, particularly cell expansion for cell and gene therapy applications. Each pod module may have a separate housing with recesses to facilitate handling and transplanting of the pod modules in stacked and non-stacked configurations near or away from the base of the bioreactor pod. One or more pod modules and associated functionality may also be combined under a unified housing. Pod modules combined under a unified housing may be stacked, arranged and / or customized for ease of use and for a particular bio-augmentation process, such as the cell and gene therapy processes described herein.

[0199] exist FIG. 29A to FIG. 29BIn the illustrated exemplary embodiment, the pod module 1004 and the pod module 1006 are the first pump module 1004 and the second pump module 1006. The first pump module 1004 and the second pump module 1006 each have a housing 1005, which each accommodates two pump heads 1013 and two pumps 1007. A total of four pumps 1007 can be any type of pump, including peristaltic pumps. One or more of the pump modules 1004 and the associated pump 1007 are fluidly connected to a liquid container in the bioreactor 1010 and the liquid container 1011 via a pipeline 1009 to pump fluid between the container 1011 and the bioreactor 1010 via a pipeline 1009. The pump module 1004 can also be connected to other containers or equipment to pump liquid to and from equipment or container pumping liquid. In other embodiments, the pump modules 1004, 1006 can each include less than two pumps and pump heads or more than two pumps and pump heads. The bioreactor pod 1000 may also include more or less than two pump modules. Each pump 1007 may also be electrically connected to a pump actuation button 1015 that can actuate the pump to flow liquid toward the container 1011 or toward the bioreactor 1010, or alternatively, a pump actuation button (e.g., having two buttons) can cause the pump 1007 to flow fluid to the left or right side of the pump 1007 and into and out of other containers or equipment depending on which of the two inputs is actuated on the pump actuation button 1015. The flow rate and direction of the fluid flow pumped by the pump modules 1004, 1006 and from the pump 1007 can be controlled by the controller 1037 based on user input at the display 1014 and the user interface and recipes stored in the memory of the controller 1037. Any type of fluid can be pumped and flowed to and from the bioreactor 1010 and the container 1011 via the pump modules 1004 and 1006 and the pump 1007, including biological fluids, cells, cell culture medium water and / or one or more biological components, fluids, solids, mixtures, solutions and suspensions, including but not limited to bacteria, fungi, algae, plant cells, animal cells, white blood cells, T cells, cell culture media, protozoa, nematodes, plasmids, viral vectors, blood, plasma, organelles, proteins, nucleic acids, lipids, plasmids, carbohydrates and / or other biological components, etc. Examples of some common biological components include E. coli, yeast, Bacillus and CHO cells. Fluids may also include cell therapy cultures and aerobic or anaerobic and adherent or non-adherent cells and microorganisms.

[0200] exist FIG. 29A to FIG. 29BIn the embodiment depicted in FIG. 1 , the pod module 1008 is a mass flow controller module 1008 (MFC module 1008) that controls the flow of fluids passing through the MFC module 1008, the conduit 1009, and reaching the bioreactor 1010. In an exemplary embodiment, the MFC module 1008 controls the flow of gas to the gas overlap assembly 1033 and the bottom sparger 1094. When the bioreactor is operated in a static mode or a dynamic mode, a gas (such as oxygen) can be directed through the gas overlap assembly 1033 and the bottom sparger 1094 and flow into the bioreactor 1010 to support a biologically active environment and amplify cell growth and proliferation in the bioreactor 1010. The MFC module 1008 and the flow rate and direction of the fluid flow (e.g., oxygen flow) passing through the MFC module 1008 and reaching the bioreactor 1010 can be controlled by the controller 1037 based on user input at the display 1014 and the user interface and recipes stored in the memory of the controller 1037. In an exemplary embodiment, the MFC module 1008 includes four gas inlets 1023 and two gas outlets 1025 fluidly connected to the conduit 1009. Two of the inlets 1023 supply gas to the gas overlap assembly 1033 through one outlet 1025 and the conduit 1009, and the other two inlets 1023 supply gas to the bottom sparger 1094 through one outlet 1025 and the conduit 1009. Other gas outlet and inlet configurations are also possible.

[0201] exist FIG. 29A to FIG. 29B In the embodiment depicted in FIG. 1 , the pod module 1017 may be an optional module integrated into the pod base 1002, or may be a separate electrical module 1017 that supplies power to components of the bioreactor pod 1000, including but not limited to the pod modules 1004, 1006, 1008, the motor 1024, the sensor 1080, and the controller 1037, via a cable 1041. The cable 1041 may transmit power and / or data to and from components of the bioreactor pod 1000, including but not limited to the pod modules 1004, 1006, 1008, the motor 1024, and the sensor 1080, and the controller 1037. The electrical module 1017 may include one or more AC power supplies, DC power supplies, distribution boxes, programmable power supplies, uninterruptible power supplies, switch mode power supplies, and / or other power supplies. The electrical module 1017 may also include an emergency stop 1019 that cuts off power and / or stops the operation of one or more components of the bioreactor pod 1000, including the pod modules 1004, 1006, 1008, the motor 1024, and the sensor 1080, and a controller 1037. In an example embodiment, the electrical module 1017 is part of the reactor pod base 1002, as previously described.

[0202] The support 1012 and pod modules 1004, 1006, 1008, 1017 may include a variety of tube 1009 and cable 1041 management clips 1027 or slots 1027 to manage, organize and arrange the tubes 1009 and cables 1041 in a manner that does not interfere with access, operation, movement or actuation of any component of the bioreactor pod 1000. The tube 1009 and cable 1041 management clips 1027 may be positioned on one or more side walls of the pod modules 1004, 1006, 1008, 1017 and one or more surfaces or platforms of the support 1012. The support 1012 may include a movable arm 1029 that is attached to the display 1014 to facilitate upward, downward and sideways movement of the display 1014 and the user interface to a position that facilitates operator input and output display.

[0203] The support 1012 may also include a scanner 1031 (such as a bar code scanner 1031) that can scan serial numbers or bar codes associated with the contents or components of the pod 1000, including serial numbers or bar codes associated with the bioreactor 1010, sensor 1080, pod modules 1004, 1006, 1008, 1017, pod base 1002, bioreactor receiver 1092, gas overlap assembly 1033, sprinkler 1094 or other components. When scanned with the scanner 1031, the barcode or serial number provides data about the component, including, but not limited to, data identifying the bioreactor 1010 type, size, impeller type, manufacturer, and date of manufacture; sensor 1080 type, calibration data, manufacturer, and date of manufacture; pod module 1004, 1006, 1008, 1017 type, capacity, calibration data, manufacturer, and date of manufacture; bioreactor receiver 1092 type, size, manufacturer, and date of manufacture; gas overlap assembly 1033 / sparger 1094 size, aperture, gas flow rate capacity, construction materials, manufacturer, and date of manufacture; and other parameters and details about the components of the pod 1000. This scanned data may be stored in the memory of the controller 1037 and used in a pod recipe, which includes a set of instructions for controlling operation, flow rates, impeller speeds, reactor operating modes (e.g., static vs. dynamic), and / or the period or length of time for each bioreactor operating mode. The pod operation recipe may be stored in the controller 1037 memory and called and run by the controller's processor based on equipment type (including but not limited to bioreactor, gas overlap assembly, sparger, impeller or sensor type and / or manufacturer) and equipment parameters (including but not limited to bioreactor volume / capacity, overhead gas flow rate capacity, sparger pore size and membrane type, impeller size and blade type) or sensor calibration data.

[0204] The controller 1037 may include one or more processors and memory for executing pod recipes in the form of machine code instructions to automate one or more components of the bioreactor 1010 and the bioreactor pod 1000. One or more bioreactor or pod automation recipes may be stored in the memory of the controller 1037 and executed by an operator via input from the display 1014 and an associated user interface. The time periods, times, and sequences in which the bioreactor 1010 is operated in dynamic and static modes during the expansion steps, as well as the associated impeller speeds and gas flow rates to the gas overlap assembly and sparger are programmable and can be stored in the controller 1037 memory in the form of recipes. The bioreactor / pod recipes may be customized, called, and executed by the controller 1037 to optimize cell growth, preservation, and recovery, particularly during isolation, activation, modification, expansion, and washing processes that pressurize the cells.

[0205] Fig.30 1 is a bioreactor pod wall 1100 according to an exemplary embodiment. The bioreactor pod wall 1100 includes two or more frames 1102 having two or more shelf sections 1104 that receive two or more bioreactor pods 1000. In other exemplary embodiments, the bioreactor pod wall 1100 may include one frame housing or include several shelves. The bioreactor pod 1000 may be about FIG. 29A to FIG. 29B The same bioreactor pod 1000 as described. An operator 1106 can interact with a terminal 1108 equipped with a display and a user interface to input and receive output from each bioreactor pod 1000, control the operation of the pod 1000, and monitor the parameters and output of the pod 1000. Each bioreactor pod 1000 in the bioreactor pod wall 1100 can be associated with and contain biological components (such as cells or transgenic cells) of a specific patient. In the example Fig.36 After the patient's cells have been expanded, modified, re-expanded, and / or treated, the cells can be reintroduced into the patient and administered as a therapeutic agent. In this manner, the bioreactor compartment wall 1100 can facilitate the administration of cell therapy to a number of patients, and each bioreactor compartment 1000 is specific, customized, and customized to a particular patient, the patient's disease and / or condition, and the patient's specific therapy.

[0206] FIG. 31A to FIG. 31D A front view, a rear view, and a cross-sectional view of a sensor assembly 1080 (or sensor group) according to an exemplary embodiment are depicted, respectively. The sensor assembly 1080 is capable of coupling, protecting, and operating sensors at the top wall or other walls (e.g., side walls) of a bioreactor disclosed herein. In an exemplary embodiment, the sensor assembly 1080 may be FIG. 29A to FIG. 29BThe sensor 1080 is connected to FIG. 29A to FIG. 29B 1060A of the bioreactor. The sensor assembly 1080 includes a sensor hub 1202 having a port connector 1204 that can be connected to the top bioreactor port disclosed herein. In an exemplary embodiment, the port connector 1204 is coupled to the port 1060A (or other bioreactor port) via a press fit, a threaded connection, a barb and cable tie connection, or other connection. The sensor assembly 1080 includes a dip tube 1206 for inserting a resistance temperature detector 1208 (or other temperature sensor) having a metal tip 1210 for measuring the temperature of the bioreactor 1010 ( FIG. 29A to FIG. 29B The temperature of the contents is shown. The sensor assembly 1080 also includes three foam sensors 1212 (or level sensors) to measure the volume, thickness or content of foam in the bioreactor 1010 and determine whether defoaming measures should be taken.

[0207] Fig.32 is a top wall 1314 (or top cap) of a bioreactor according to an example embodiment. Top wall 1314 can be any of the top walls of the bioreactors disclosed herein (e.g., top walls 14, 368, 514, 714, 1026). In an example embodiment, top wall 1314 can be about FIG. 29A to FIG. 29B The top wall 1026 of the bioreactor 1010 is depicted. Fig.32 The top end wall 1314 depicted in FIG. 1 may include any combination of top end wall 14, 368, 514, 714, 1026 components, ports, sensors, gas overlap components, and other components. Fig.32 In the embodiment, the top end wall 1314 has four corners 1301-1304, each having a radius of curvature r, r, r, and R. Three of the corners 1301-1303 have a first radius of curvature r, and the fourth corner 1304 has a second radius of curvature R that is different from the other three corners 1301-1304. In the exemplary embodiment, the fourth corner 1340 has a larger radius of curvature R than the other three corners 1301-1304 to form a foolproof feature that ensures that the top end wall 1314 is installed in the housing of the bioreactor (e.g., bioreactor 1010) in the correct orientation / with the housing of the bioreactor. The housing of the bioreactor disclosed herein may also have the same shape as the cap, wherein the radius of curvature of the three corners is smaller than the radius of curvature of the fourth corner of the housing to correctly assemble and orient the top end wall 1314 into the housing.

[0208] FIG. 33A to FIG. 33EA series of impellers 1401-1405 are depicted, respectively, which can be coupled to any of the bioreactors disclosed herein to mix the contents of the bioreactor. Impellers 1401-1405 are rotatably and movably disposed within a compartment of the bioreactor to mechanically mix biological components and other fluids within the compartment when the bioreactor is operated in a dynamic mode. In an exemplary embodiment, any of the impellers 1401-1405 can be interchangeably mounted to a bioreactor disclosed herein via any of the impeller mounting hubs disclosed herein (e.g., impeller mounting hubs 540, 740, 1022, 1500). In an exemplary embodiment, any of the impellers 1401-1405 can be interchangeably mounted to a bioreactor disclosed herein. FIG. 29A to FIG. 29B The mounting hub 1022 of the described bioreactor 1010. Each impeller 1401-1405 has a main drive shaft 1406A-1406E. Each impeller 1401-1405 also includes a top mounting portion 1414A-1414E that is coupled to the impeller mounting hub (e.g., impeller mounting hub 540, 740, 1022, 1500) to facilitate engagement with the top wall of any of the disclosed bioreactors and motors described herein. Each impeller 1401-1405 has three sets of impeller blades 1408-1412.

[0209] The impeller 1401 has two supports 1407 radially spaced from the main shaft 1406A, which can increase stability during mixing. The impeller 1401 also includes three sets of three blades (three-leaf blades) 1408A-1408C. The three-leaf blades 1408A and 1408C are spaced the same distance as the three-leaf blade 1408B and are connected to the supports 1407 and the main shaft 1406A at three different sections of the shaft 1406A.

[0210] Impeller 1402 includes three sets of three-blade blades 1409A-1409C. Three-blade blades 1409A and 1409C are spaced the same distance as three-blade blade 1409B and are coupled to main shaft 1406B and grouped near the bottom section of shaft 1406B.

[0211] Impeller 1403 includes three groups of two blades (two-leaf blades) 1410A-1410C. Two-leaf blades 1410A and 1410C are spaced the same distance as two-leaf blade 1410B and are coupled to main shaft 1406C and grouped near the bottom section of shaft 1406C, but first two-leaf blade 1410A is positioned and coupled on main shaft 1406C at a higher position than three-leaf blade 1409A on main shaft 1406B of impeller 1402.

[0212] Impeller 1404 includes three groups of three-leaf blades 1411A-1411C. The distance between three-leaf blades 1411A and 1411B is greater than the distance between three-leaf blades 1411B and 1411C, wherein three-leaf blades 1411B and 1411C are coupled to main shaft 1406D and grouped further toward the bottom section of shaft 1406D.

[0213] Impeller 1405 includes three groups of three-leaf blades 1412A-1412C. The distance between three-leaf blades 1412A and 1412B is greater than the distance between three-leaf blades 1412B and 1412C, wherein three-leaf blades 1412B and 1412C are coupled to main shaft 1406E and are grouped further toward the bottom section of shaft 1406E. First three-leaf blade 1412A is positioned and coupled to main shaft 1406E at a higher position than first groups of blades 1409A-1411A on main shafts 1406B-D of impellers 1402-1404, respectively.

[0214] In an exemplary embodiment, the impellers 1401-1405 described herein include one to five groups of blades (1 to 5 blade groups), and each blade group may each have two to four blades (two-leaf blades, three-leaf blades, or four-leaf blades). FIG. 33A to FIG. 33E , the blades may have a pitch of 0° to 45° with a horizontal plane perpendicular to the vertical axis (e.g., long axis) of the main drive shaft 1406A-1406E. Each blade set can be coupled to the main drive shaft 1406A-1406E at the same distance interval or at different distance intervals. In an example embodiment, the blade sets (e.g., 1408A-1412C) are spaced 5 mm to 50 mm apart between each blade set and coupled to the main drive shaft 1406A-1406E. The spacing between the blade sets can be customized to achieve optimal mixing and cell growth.

[0215] FIG. 34A to FIG. 34CA perspective view, a cross-sectional view, and a partial top view of an impeller mounting hub 1500 according to an example embodiment are depicted, respectively. The impeller mounting hub 1500 includes a mounting shaft 1502 that extends and protrudes through the impeller mounting hub 1500. The mounting shaft 1502 has a bottom mounting portion 1506 that can be mounted and coupled to a drive shaft 1504 of an impeller in a compartment of a bioreactor (e.g., 510, 710, 1010) disclosed herein. The bottom mounting portion 1506 of the impeller mounting hub 1500 is coupled to a top mounting portion 1516 of a drive shaft 1504 or other shaft (e.g., drive shaft 616, 716, 1018) of an impeller assembly (e.g., impeller assembly 550, 750, 1016) disclosed herein via a press fit or other mechanical connection. In an example embodiment, the bottom mounting portion 1506 of the impeller mounting hub 1500 may include structures, protrusions, extensions, or pins 1518 that are keyed to mate (e.g., press fit) with and couple to recesses 1520 in the top mounting portion 1516 of the drive shaft 1504. The mounting hub 1500 can be coupled to the top mounting portions of other drive shafts disclosed herein (e.g., drive shafts 616, 716, 1018) in a similar keyed manner. A portion of the mounting shaft 1502 engages with one or more bearing assemblies 1514A, 1514B to facilitate rotation of the mounting shaft 1502. A portion of the mounting shaft 1502 and the one or more bearing assemblies 1514A, 1514B are received and sealed within a hub cavity 1512 of the mounting hub 1500. The mounting hub 1500 may include a threaded portion 1508 to engage and couple to the top wall of the bioreactor and the threaded bearing ports disclosed herein (e.g., threaded bearing ports 530, 730, 1030). A dust cover 1510 is located near or adjacent to the terminal end of the impeller mounting hub 1500 to accommodate the bearings 1514A-1514B and prevent dust or particles from entering the hub cavity 1512.

[0216] FIG. 35A to FIG. 35B A perspective view and a cross-sectional view of an impeller assembly 1600 according to an example embodiment are depicted, respectively. The impeller assembly 1600 includes an impeller mounting hub 1602, a main drive shaft 1606 coupled to the impeller mounting hub 1602, two supports 1608A-1608B, and three sets of three-leaf blades 1610A-1610C coupled to the main drive shaft 1606, and supports 1608A-1608B at three different sections along the shaft 1606. The three-leaf blades 1610A-1610C can be spaced, grouped, and configured in a variety of ways, such as with respect to FIG. 33A to FIG. 33EThe main drive shaft 1606 may include two flexible, actuatable or bendable clips or prongs 1612A-1612B that may engage or snap fit within the hub receiver 1604 of the impeller mounting hub 1602 to removably attach the shaft 1606 to the impeller mounting hub 1602. When the prongs 1612A-1612B snap fit or press fit within the narrow portion 1615 of the hub receiver 1604, the prongs elastically bend, actuate or compress inwardly to a compressed position, and when the prongs 1612A-1612B snap fit or press fit through the wider portion 1617 of the hub receiver 1604, the prongs actuate, expand or spring back to the expanded position like a spring. The flanges 1614 on the prongs 1612A-1612B prevent the prongs 1612A-1612B from being pulled through the narrower portion or channel 1615 of the impeller mounting hub 1602 unless a threshold force is applied or used to pull the prongs 1612A-1612B / drive shaft 1606 out of the hub receiver 1604. In other embodiments, the hub receiver 1604 does not have the narrower portion 1615 and wider portion 1617, and the prongs 1612A-1612B are simply compressed (or clamped) to a compressed position to fit the prongs into the hub receiver 1604, and allowed to resiliently spring back to an expanded position to retain the prongs 1612A-1612B and drive shaft 1606 in the hub receiver 1604. In this manner, the prongs 1612A- 1612B may quickly facilitate attachment and removal of the drive shaft 1606 (as well as other drive shafts disclosed herein) from the impeller mounting hub 1602 .

[0217] Fig.36An autologous cell therapy system and process flow 1700 is depicted according to an example embodiment, including one or more bioreactors or bioreactor pods 1710 operating in a static mode and / or a dynamic mode. The autologous cell therapy system 1700 includes one or more equipment modules, including a blood processing system 1704 disclosed herein, a cell and bead processing system 1706, a gene editing system 1708 (e.g., an electroporation system), a bioreactor / bioreactor pod 1710 (e.g., bioreactor pod 1000 and bioreactor 1010), an incubator 1712, a refrigerator 1714, and associated automation software for controlling each equipment module with a controller 1716. The autologous cell therapy system 1700 depicts one patient 1702 and includes three blood processing systems 1704 and two bioreactors / pods 1710, one gene editing system 1708, one incubator 1712, and one refrigerator 1714. The three blood processing systems 1704 and two bioreactors / pods 1710 shown can be the same system / bioreactors where cells and media flow and recirculate, or separate systems / bioreactors.Any combination of equipment modules described herein can be combined and customized to meet operator and patient needs.

[0218] In an exemplary embodiment, the autologous cell therapy system 1700 is dedicated to one patient 1702 and includes three blood processing systems 1704 and two bioreactors / pods 1710, a cell and bead processing system 1706, an electroporation system 1708, and a freezer 1714. The bioreactors / pods 1710 can be operated as incubators using one or more top or bottom spargers, so if a dual-mode bioreactor 1710 is implemented, the incubator 1712 is not necessary.

[0219] At step 1, the cell therapy process begins by drawing a blood sample from a patient 1702. The blood sample includes plasma, red blood cells, platelets, and white blood cells or leukocytes.

[0220] At step 2 of the cell therapy process, the blood sample is flowed or supplied to a blood processing system 1704, which can be used to separate white blood cells (leukocytes) from the rest of the patient's blood components. Example blood processing systems 1704 that can be used in the cell therapy system include Gibco TM CTS TM Rotea TMCountercurrent centrifugal system and blood processing system and method disclosed in WO2018 / 204992, which is incorporated herein by reference in its entirety. The blood processing system 1704 may include a centrifuge (e.g., a countercurrent centrifuge) or other equipment for separating white blood cells (leukocytes) from the rest of the patient's blood components during leukocyte removal. The separated leukocytes may also be washed, reconstructed and / or suspended in fresh cell culture medium or other culture medium at the blood processing system 1704, and then an amplification step is performed at the bioreactor / cabin 1710 or an isolation / activation step is performed at the cell and bead processing system 1706.

[0221] At step 3 of the cell therapy process, the separated white blood cells are processed in a cell and bead processing system 1706, which includes at least a magnet and magnetic beads for processing the cells. Exemplary cell and bead processing systems 1706 that can be used in the cell therapy system include Gibco TM CTS TM DynaCellect TM Magnetic separation system and bead processing system, method, equipment and processing workflow disclosed in WO2022 / 081519, which is incorporated herein by reference in its entirety. Cell and bead processing system 1706 can be used to bind magnetic beads to specific cell types (e.g., stem cells, general leukocytes, granulocytes, monocytes, total T cells, helper T helper cells, regulatory T cells, cytotoxic T cells, B cells, natural killer cells, platelets, etc.), isolate, activate and wash bound or unbound cells. For example, magnetic beads can be bound to target cell types via antibodies between beads and cells, and the antibodies are bound to the surface receptors of cells through the antigen binding sites of antibodies. The specific region of the antibody (e.g., the Fc region of the antibody) is then connected to a joint, which connects the antibody to the magnetic beads. Magnets or magnet systems can be used to attract and separate magnetic beads via the lysis mechanism described in detail in WO2022 / 081519 in the case of cell binding or after the cells are released from the magnetic beads, which is incorporated herein by reference in its entirety.

[0222] In both the positive cell isolation process and the negative cell isolation process, the target cells can be bound to magnetic beads, isolated, and activated within a bag or container of the cell and bead handling system 1706, as described in detail in WO2022 / 081519, which is incorporated herein by reference in its entirety. Example commercially available magnetic beads that can be used to isolate and activate target cells include, but are not limited to, DYNABEADS TM HumanT-Expander CD3 / CD28 (Thermo Fisher Scientific, catalog number 11141D), CTS TM DYNABEADS TMCD3 / CD28 (Thermo Fisher Scientific, catalog number 40203D), CTS TM DYNABEADS TM TregXpander (Thermo Fisher Scientific, catalog number 46000D). During the positive cell isolation process, magnetic beads are bound to target cells, and the beads / cell complexes are pulled to the magnets in the cell and bead processing system 1706. The supernatant is discarded, and the beads / cell complexes are washed with enzyme lysis or other lysis mechanisms (described in detail in WO2022 / 081519, which is incorporated herein by reference in its entirety) to produce activated target cells. In negative cell isolation, magnetic beads are bound to all unwanted cells or non-target cells, and non-target cells are attracted to the magnets in the blood processing system 1704 to exhaust all unwanted cells and keep unbound target cells in the separation bag.

[0223] Alternatively, at step 3 of the cell therapy process, the leukocytes may undergo cell propagation and / or expansion in one or more cell expansion processes in a bioreactor / cartridge 1710 (e.g., bioreactor cartridge 1000 and reactor 1010) operating in static mode, dynamic mode, or both for a predetermined and / or alternating period of time, and then be isolated / activated at the cell and bead handling system 1706. Operating in both static mode and dynamic mode (e.g., alternating periods of static mode and dynamic mode) during cell expansion may reduce strain on the cells, promote optimal cell growth, and preserve sensitive cells undergoing washing, isolation, activation, and / or modification that cause cell stress and death during the cell therapy process 1700.

[0224] At step 4 of the cell therapy process, the target cells harvested from the cell and bead processing system 1706 may be washed, reconstituted, and / or suspended in fresh cell culture medium or other culture medium and liquid (e.g., freezing medium, water, buffer) at the bead processing system 1706, and then undergo an expansion step at the bioreactor / cartridge 1710. The target cells may also be supplied to the blood processing system 1704, where they are separated, washed, reconstituted, and / or suspended in fresh cell culture medium or other culture medium and liquid, and then undergo an expansion step at the bioreactor / cartridge 1710. This step 4 may also be omitted.

[0225] At step 5, target cells isolated and / or activated in the cell and bead handling system 1706 in step 3 and washed, reconstituted and / or suspended in fresh cell culture medium in step 4 undergo cell propagation and / or expansion for predetermined and / or alternating time periods in one or more cell expansion processes in a bioreactor / bioreactor chamber 1710 (e.g., bioreactor chamber 1000 and reactor 1010) operated in static mode, dynamic mode, or both modes to promote optimal cell growth and preserve sensitive cells that undergo washing, isolation, activation and / or modification steps (subjecting the cells to stress).

[0226] At step 6 of the cell therapy process, the target cells may be flowed, fed or transferred to a gene editing system 1708 that edits, modifies or inserts target DNA, RNA, protein and / or other molecules into the target cells to produce a therapeutic outcome. Exemplary gene editing systems 1708 that may be used in the cell therapy system 1700 include CTS TM Xenon TM Electroporation system, Neon TM NxOB TM Electroporation systems, and gene editing systems, methods, equipment, and processing workflows disclosed in U.S. Publication Nos. 2021123009, 20230110090, and U.S. Patent No. D965170, which are incorporated herein by reference in their entirety.

[0227] At step 7 of the cell therapy process, the modified cells edited in the gene editing system 1708 can be flowed, supplied or transferred to the blood processing system 1704, where the modified cells are separated, washed, reconstituted and / or suspended in fresh cell culture medium or other culture medium and liquid. This step 7 can also be omitted.

[0228] At step 8 of the cell therapy process, the cells modified in the gene editing system 1708 in step 6 may be flowed, fed or transferred to a bioreactor / bioreactor capsule 1710 to undergo cell propagation and / or expansion for predetermined and / or alternating time periods within a bioreactor / bioreactor capsule 1710 (e.g., bioreactor capsule 1000 and reactor 1010) operating in a static mode, a dynamic mode, or both modes to promote optimal cell growth and preservation of cells undergoing washing, isolation, activation and / or modification steps. Operating the bioreactor 1710 in alternating periods of static and dynamic modes during cell expansion can reduce strain on the cells and promote optimal cell growth and preservation during the cell therapy process 1700.

[0229] In optional step 9, the modified cells expanded in the bioreactor / bioreactor pod 1710 can be flowed, supplied or transferred to an incubator 1712 for temporary storage or further expansion. In an exemplary embodiment, implementation of the bioreactor pod or dual-mode bioreactor 1710 eliminates the need for an incubator due to the operability of the bioreactor / bioreactor pod 1710 in static mode and oxygenation of the cells from the gas permeable membrane, sidewalls, top gas overlap assembly and / or sparger disclosed herein. The bioreactor 1710 can also be placed inside the incubator 1712, where gas is supplied through the sidewall gas permeable membrane to further expand the cells, while operating in static mode without impeller mixing, as previously described.

[0230] At step 10 of the cell therapy process, the modified cells expanded in the bioreactor cartridge / bioreactor cartridge 1710 may be flowed, supplied or transferred to the blood processing system 1704, in which the modified cells and expanded cells are separated, washed, reconstituted and / or suspended in fresh cell culture medium or frozen culture medium in preparation for cold chain storage and processing and / or injection into the patient 1702 as a therapeutic agent.

[0231] At step 11 of the cell therapy process, the washed, reconstituted and / or suspended modified cells can be flowed, supplied or transferred to a freezer 1714 along with freezing medium and in a freezing bag for freezing, transport and ultimately thawing and the modified cells administered to a patient 1702 as a therapeutic agent.

[0232] The controller 1716 may include one or more processors, memory, and software instructions executed by the processor to automate the cell therapy system, process 1700, and associated equipment modules, including one or more blood processing systems 1704, cell and bead processing systems 1706, gene editing systems 1708 (e.g., electroporation systems), bioreactors / bioreactor pods 1710, incubators 1712, freezers 1714, and associated sensors and support modules. The controller may also include a client computer, a display, and a user interface with operator input and system output for controlling the cell therapy system / process 1700. One or more equipment and process automation recipes may be stored in the memory of the controller 1716 and run by the operator via the client computer and the user interface. The time period, number of times, and sequence of the bioreactor / pod 1710 operating in dynamic mode and static mode during the amplification step and process are programmable and may be stored in the controller 1716 memory in the form of a recipe. The recipe may be customized, called, and run by the controller 1716 to optimize cell growth, preservation, and recovery.

[0233] The bioreactor disclosed herein has many unique advantages. For example, the bioreactor can realize the static culture of cells (i.e., without mixing) and the dynamic growth of cells (i.e., light, heavy, low RPM, high RPM, intermittent or continuous mixing) in the same bioreactor, thereby minimizing the delay, waste and danger associated with transferring cells between bioreactors or other equipment. The uniqueness of the bioreactor is that they are configured to optimize the production under both the static operation mode and the dynamic operation mode according to the density, state, sensitivity and application of cells in the cell culture. In addition, when in static mode (when the mixer / impeller is not operating) and dynamic mode (when the mixer / impeller is operating), the bioreactor is easy to rotate into a horizontal orientation, a vertical orientation or an angled orientation to further promote optimal cell growth. The dual-mode bioreactor disclosed herein can be rotated to different orientations to expand during cell expansion and during dynamic mode and static mode, while the bioreactor moves via rotation or translation. The uniqueness of the bioreactor is that relatively cheap parts that contact the suspension during processing can be discarded / recycled after a single use, while relatively expensive parts can be reused without any sterilization. There are other benefits and unique features as well.

[0234] Any number of different cell culture media and culture medium components can be used in combination with the bioreactor provided herein. In many examples, cell culture media and culture medium components will change with the use of the cultured cells and the purpose of cell expansion (e.g., use of expanded cells, protein production, antibody production, etc.).

[0235] Cells (e.g., animal cells such as mammalian cells) that can be expanded using the devices (e.g., bioreactors) and methods described herein include immortalized cells (e.g., hybridoma cells) and primary cells (e.g., T cells, B cells, hepatocytes, etc.). Some types of cells that can be expanded using the devices and methods described herein include stem cells (e.g., induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, etc.). Other types of cells that can be expanded using the devices and methods described herein include immune system cells, such as T cells (e.g., CD4+T cells, CD8+T cells, regulatory T cells, Th17T cells, γδT cells, memory T cells (e.g., central memory T cells), natural killer T cells, mucosa-associated invariant T cells, etc.), natural killer (NK) cells, B cells, dendritic cells, antigen presenting cells, etc.

[0236] Some specific examples of cells that can be expanded using the devices and methods described herein include African green monkey cells (e.g., BSC cells), HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, CHO cells, CHO-K1 cells, NS-1 cells, MRC-5 cells, WI-38 cells, 3T3 cells, 293 cells, Per.C6 cells, and chicken embryo cells. In some instances, one or more CHO cell variants (e.g., CHO-K1) in a CHO cell line or several specific CHO cell variants optimized for large-scale protein production are expanded.

[0237] For example, T cells can be expanded in many different culture media, including X-VIVO 15 TM (Lonza, catalog number BE02-060Q) and OPTMIZER TM CTS TM SFM, AIM-V and RPMI 1640 (Thermo Fisher Scientific, catalog numbers A1048501, 0870112DK, 11875119). In addition, T cells can be expanded in the presence or absence of serum. Additionally, T cells can be expanded with serum replacements, such as CTS TM Immune Cell Serum Replacement (ICSR) (Thermo Fisher Scientific, catalog number A2596101).

[0238] T cells can be activated before, during and / or after amplification. For example, T cells can be activated in a bioreactor during amplification. By further example, T cells can be activated by contacting with anti-CD3 and anti-CD28 antibodies. Such antibodies can be bound to one or more solid supports (e.g., beads). In addition, T cells can also be contacted with one or more cytokines (e.g., interleukin-2, etc.) before, during and / or after amplification. Therefore, a method for amplifying T cells in a bioreactor is provided herein.

[0239] Culture media that can be used in conjunction with the devices and methods described herein include Eagle's MEM (minimum essential medium), Ham's F12, F-12K, Dulbecco's, Dulbecco's modified Eagle's medium, DMEM / Ham's F12 1:1, Trowell's T8, A2, Waymouth, Williams E, MCDB 104 / 110, RPMI-1640 medium, RPMI-1641 medium, Iscove's modified Dulbecco's medium, McCoy's 5A, Leibovitz's L-15, EX-CELL TM Series 300 (JRH Biosciences, Lenexa, KS), protamine-zinc-insulin medium. The medium may contain serum or be serum-free.

[0240] Cells can be expanded in a "fed-batch cell culture" process. "Fed-batch culture" refers to a batch culture in which animal cells and culture medium are initially supplied to a culture vessel, and additional culture nutrients are supplied to the culture continuously or in discrete increments during the culture, with or without periodic cell and / or product harvesting prior to termination of the culture. Fed-batch culture includes "semi-continuous fed-batch culture," in which the entire culture (which may include cells and culture medium) is periodically removed and replaced with fresh culture medium.

[0241] Fed-batch culture differs from simple "batch culture" in that all components of the cell culture (including animal cells and all culture nutrients) are supplied to the culture vessel at the beginning of the culture process.

[0242] Cells can also be expanded in a perfusion process. In perfusion culture, cells are confined in culture by (eg, filtration), and culture medium is continuously or intermittently introduced into and removed from the culture vessel.

[0243] Some aspects of the compositions and methods described herein relate to obtaining oxygen and removing carbon dioxide from the cells of amplification. It is generally expected that these cells are easy to obtain oxygen when carbon dioxide is effectively removed. According to these ideas, the cells of amplification as described herein will be present in a bioreactor, wherein one or two parameters of these parameters can be coordinated to achieve effective cell amplification. The O2 concentration in such bioreactors can be between 15% and 25% (e.g., about 15% to about 24%, about 17% to about 25%, about 18% to about 25%, about 20% to about 25%, about 22% to about 25%, about 23% to about 25%, etc.). Further, the CO2 concentration in such bioreactors can be between 2% and 7%.

[0244] In many instances, as previously discussed, gas exchange is facilitated by using a gas permeable membrane in contact with the culture medium. Such membranes may be located on one or more sides, top and / or bottom of the bioreactor and allow O2 to enter the culture medium and allow CO2 to leave the culture medium. In some instances, the gas permeable membrane used in the bioreactor and methods described herein may be composed of or may include a gas permeable silicone (e.g., dimethyl silicone), and / or the thickness of the gas permeable membrane may be between 0.001 inch and 0.01 inch (e.g., about 0.005 inch to about 0.007 inch, about 0.002 inch to about 0.007 inch, about 0.003 inch to about 0.007 inch, about 0.005 inch to about 0.009 inch, about 0.004 inch to about 0.008 inch, etc.).

[0245] In some instances, it may be desirable to have a glutamine source in the culture medium. In this case, the glutamine source can be a glutamine source that will not form a large amount of ammonia. An example of this glutamine source is L-alanyl-L-glutamine dipeptide. When present, this glutamine reagent can be present in a concentration between about 1mM to about 20mM (e.g., about 2mM to about 20mM, about 5mM to about 18mM, about 10mM to about 20mM, about 8mM to about 27mM, etc.).

[0246] Furthermore, while the incubation temperature for expansion of cells including immune cells (eg, NK cells, T cells, B cells and / or APCs) can vary, mammalian cells are typically cultured at a temperature between 34°C and 40°C, such as 37°C.

[0247] Depending on the type of cells being expanded, the expanding cells can be contacted with one or more chemokines or cytokines. Chemokines and cytokines that can be used include interleukin-1α, interleukin-2, interleukin-4, interleukin-1β, interleukin-6, interleukin-12, interleukin-15, interleukin-18, interleukin-21, and transforming growth factor β1.

[0248] The bioreactors described herein can also be used to incubate cells in combination with one or more processes associated with inducing materials (e.g., DNA, RNA, proteins, proteins / nucleic acids / complexes, etc.) into cells (e.g., eukaryotic cells such as mammalian cells). Material introduction processes include transduction (e.g., viral transduction) and transfection. Exemplary transduction and transfection methods include calcium phosphate transfection, liposome transfection, nucleofection, sonoporation, transfection by heat shock, magnetization transfection, and electroporation. Vectors used in exemplary viral transduction methods that can be used for the methods described herein include, but are not limited to, retroviruses (e.g., lentiviruses), adenoviruses, and adeno-associated virus vectors.

[0249] The method of introducing the material will vary depending on many factors, including the material to be introduced into the cell. For example, electroporation is generally more suitable for introducing the guide RNA / Cas9 complex into the cell than lentiviral transduction. In addition, when attempting to introduce a nucleic acid (e.g., a nucleic acid encoding a chimeric antigen receptor) into a cell and wish to maintain high cell viability, viral transduction may be more suitable than electroporation.

[0250] Electroporation is a non-viral process that can be used to introduce a variety of materials into cells. Electroporation involves the application of an electric field to cells, resulting in damage to the cell membrane, thereby allowing cellular uptake delivery of exogenous materials.

[0251] There has been a great deal of work done on the mechanistic theory associated with the response of cell membranes to electric field pulses, which rapidly increase the transmembrane voltage Um(t) of the cell membrane to a value at which the cell membrane porosity rises dramatically (see Weaver et al., Bioelectrochemistry 87:236-243 (2012)). The change in membrane porosity is thought to be caused by pore formation. Thus, material uptake is thought to be mediated by inducing pore formation in the cell membrane.

[0252] The large electric field pulses used for electroporation can kill cells by heating or not heating is the main reason. Two non-thermal killing mechanisms are believed to be through induction of apoptosis or necrosis. Further, it is believed that high-intensity electric field cell killing is more through apoptosis, while low-intensity electric field cell killing is believed to be more through necrosis. Therefore, regardless of the cell death mechanism, it is usually necessary to adjust the electric field conditions and other parameters so as to maintain high cell viability.

[0253] After the cells are exposed to an electric field (e.g., electroporation), the bioreactors described herein can be used to maintain the viability of the cells by operating in a static mode or in a dynamic mode at a low mixing RPM (e.g., less than 40 RPM or less than 10 RPM). Exemplary methods of maintaining cell viability include exposing the cells to an electric field and then incubating the cells while operating the bioreactor described herein in a static mode. Such incubation can be performed in a culture medium or a culture medium designed to allow the cells to remain in a low metabolic state during incubation (e.g., an osmotic stabilizing solution containing minimal nutrients sufficient to prevent a significant decrease in cell viability).

[0254] In many examples, the cells are incubated in a bioreactor for a fixed incubation period (e.g., about 30 minutes to about 21 days, about 30 minutes to about 3 hours, about 30 minutes to about 5 hours, about 30 minutes to about 10 hours, about 30 minutes to about 15 hours, about 30 minutes to about 20 hours, about 30 minutes to about 24 hours, about 30 minutes to about 40 hours, about 1 hour to about 5 hours, about 1 hour to about 10 hours, about 1 hour to about 24 hours, about 5 hours to about 15 hours, about 5 hours to about 24 hours, about 10 hours to about 30 hours, about 24 hours to about 21 days, about 2 days to about 21 days, about 5 days to about 21, about 8 days to about 21 days, about 24 hours to about 48 hours, about 24 hours to about 72 hours, etc.) after electroporation, wherein no mechanical mixing occurs (static mode) or A low level of mechanical mixing (impeller RPM in dynamic mode is less than or equal to 10 RPM (e.g., about 0.1 RPM to about 10 RPM, about 0.5 RPM to about 10 RPM, about 0.8 RPM to about 10 RPM, about 1.0 RPM to about 10 RPM, about 2.0 RPM to about 10 RPM, about 3.0 RPM to about 10 RPM, about 4.0 RPM to about 10 RPM, about 5.0 RPM to about 10 RPM, about 0.1 RPM to about 1 RPM, about 0.1 RPM to about 0.8 RPM, about 0.1 RPM to about 0.6 RPM, about 0.5 RPM to about 1 RPM, about 0.3 RPM to about 1 RPM, about 0.3 RPM to about 0.8 RPM, etc.)) to allow the cells to recover from the effect of electroporation, referred to herein as the "recovery incubation period."

[0255] The recovery incubation period may alternate between static mode and dynamic mode. For example, the bioreactor may be operated in static mode for a period of time and then in dynamic mode for a period of time. An exemplary set of conditions is 30 minutes in static mode, followed by 30 minutes in dynamic mode, with an impeller RPM of 2 in dynamic mode. Using the above set of exemplary conditions for illustration, the ratio of static mode to dynamic mode will be 1:1. In some instances, the ratio of static mode to dynamic mode may be 1:10 to 10:1 (e.g., 1:1 to 1:10, 1:1 to 1:5, 1:1 to 1:3, 10:1 to 1:1, 1:10 to 5:1, 10:1 to 3:1, 1:5 to 5:1, 1:2 to 2:1, etc.).

[0256] Additionally, the number of alternations between static mode and dynamic mode (or mixed "pulses") during the recovery period or the entire post-electroporation culture period can be 1 to 1,000 (e.g., about 10 to about 1,000, about 20 to about 1,000, about 100 to about 1,000, about 10 to about 500, about 10 to about 250, about 10 to about 150, about 30 to about 250, about 50 to about 500, etc.).

[0257] Additionally, O2 and CO2 concentrations in the bioreactor can be adjusted during the recovery incubation period to maintain high cell viability.

[0258] Cells (e.g., mammalian cells) can be incubated in a bioreactor as described herein before, after, and / or during exposure to an electric field (e.g., electroporation). For example, cells can be cultured in a bioreactor as described herein, then removed from the bioreactor, and then reintroduced into the same or a different bioreactor. As an alternative to the above, cells can be electroporated within a bioreactor. In addition, cells (e.g., T cells) can be obtained from a patient, electroporated, and then introduced into a bioreactor as described for culture. These cultured T cells can then be reintroduced into the patient.

[0259] One method of increasing the viability of cells exposed to an electric field (e.g., electroporation) is by pre-incubating the cells with high-density lipoprotein (HDL). For example, prior to electroporation, mammalian cells (e.g., T cells) can be incubated in a CTSOPTMIZER TM(Thermo Fisher Scientific, catalog number A37050-01) with 6 mg / l HDL for three days. Methods such as these are described in U.S. Patent Publication No. 2021 / 0024882, entitled "COMPOSITIONS AND METHODS FOR ENHANCING CELLCULTURE," published on January 28, 2021, the entire disclosure of which is incorporated herein by reference. In addition, such pre-incubation can be performed in a bioreactor described herein. Thus, provided herein are methods of culturing cells with HDL for a period of time (e.g., from about 1 day to about 5 days, from about 2 days to about 4 days, etc.), and then introducing one or more materials into these cells.

[0260] Thus, the methods described herein include those methods that include introducing materials into cells by electroporation, followed by incubation of the cells in a bioreactor as described herein in a static mode, a dynamic mode, and / or an alternating mode of cell expansion, storage, and processing within a single bioreactor. In addition, such methods may involve pre-incubation of the cells with HDL.

[0261] Cells can be transduced with viral vectors in the bioreactors described herein. Such transduction methods include contacting these cells in the bioreactor with one or more viral vectors. In many examples, these viral vectors will contain nucleic acids including nucleic acid regions for inserting intracellular nucleic acids (e.g., chromosomes of cells introduced into nucleic acid regions) and / or encoding proteins for intracellular expression (e.g., Cas9 proteins, chimeric antigen receptors (CAR) etc.).

[0262] Provided herein are methods for culturing cells, introducing materials into cells and / or transforming cells. By way of example, activated T cells can be amplified in a bioreactor as described herein. Then, under conditions that allow lentiviral vectors to enter T cells to produce CAR-T cells, lentiviral vectors encoding CARs can be introduced into a bioreactor. Then, CAR-T cells can be further amplified in a bioreactor.

[0263] In many examples, viral (e.g., lentiviral) transduction will occur in a bioreactor as described herein for a period of time (e.g., about 1 hour to about 2 days, about 3 hours to about 1 day, about 5 hours to about 2 days, etc.) without mechanical mixing (static mode) or with minimal mechanical mixing (equal to or less than 10 impeller RPM in dynamic mode). The resulting transduced T cells can then be placed under recovery incubation conditions similar to the electroporation conditions described above. In addition, during the recovery incubation period, viral particles can be removed from the bioreactor.

[0264] Example 1: Conditions for culturing T cells

[0265] T cell isolation: using Dynabeads TM Untouched TM The Human T Cell Kit (Thermo Fisher Scientific, Catalog No. 11344D) negatively isolates primary human T cells from normal donors from PBMCs. The kit can be used to remove cells with the following markers: CD14, CD16 (a and b), CD19, CD36, CD56, CD123, and CD235A (e.g., B cells, NK cells, monocytes, platelets, dendritic cells, granulocytes, and erythrocytes).

[0266] Medium: Basic growth medium containing X-VIVO 15 TM (Lonza, catalog number BE02-060Q), OpTmizer TM CTS TM SFM, AIM-V SFM and RPMI 1640 (Thermo Fisher Scientific, catalog number A1048501, 0870112DK, 11875119) were used for T cell expansion. The culture medium was supplemented with 5% human AB serum (hABs) (Gemini Bio-Products) or 2.5% CTS immune cell serum replacement (Thermo Fisher Scientific, catalog number A2596101).

[0267] Activation: Using Dynabeads TM Human T-Expander CD3 / CD28 (Thermo Fisher Scientific, Catalog No. 11141D) was used to activate T cells at a ratio of 3 beads per T cell in the presence of 100 IU / ml of rIL-2 (Thermo Fisher Scientific, Catalog No. PHC0021).

[0268] Expansion: Maintain T cells at 5×10 6 Cells / ml were counted on the 3rd, 5th, 7th and 10th days using a Beckman-Coulter Vi-Cell analyzer. In addition, rIL-2 was supplemented on these same days. Cell growth is expressed as the expansion times over time. The culture medium was replaced on the 5th and 7th days, and 100 IU / ml of rIL-2 was supplemented on the 3rd, 5th and 7th days. The culture temperature was 37°C. The CO2 concentration of the culture medium was maintained at about 5%. The O2 concentration of the culture medium was maintained at about 17% to 21%.

[0269] Endpoints: Cell phenotype was assessed on day 10 by staining T cells with anti-CD3-Pacific Orange, anti-CD4-FITC, anti-CD8-Pacific Blue, anti-CD62L-APC, and anti-CCR7-PE (Thermo Fisher Scientific, catalog numbers CD0330, 11-0041-82, MHCD0828, 17-0621-82, 12-1971-82). Dynabeads were removed from the cultures on day 10 to assess cytokine production (data not shown). TM Human T-amplifier CD3 / CD28, T cells were washed and placed in fresh medium overnight. 2.5 million T cells were cultured at 1×10 6 T cells / mL were seeded and restimulated with human T-amplifier CD3 / CD28 at a 1:1 beads to cells ratio and incubated for 24 hours. The supernatant was collected and stained with LUMINEX TM INVITROGEN TM Human cytokine magnetic bead 35-link detection panel (Thermo Fisher Scientific, catalog number LHC6005M) was processed for analysis.

[0270] The present invention may be embodied in other specific forms without departing from the essence or essential characteristics of the present invention. The described embodiments should be considered in all respects as merely illustrative and not restrictive. Therefore, the scope of the present invention is indicated by the appended claims rather than by the above description. All changes within the meaning and equivalent range of the claims will be included in their scope.

Claims

1. A bioreactor, comprising: a housing comprising a plurality of walls defining a compartment adapted to hold a liquid, a selected one of the plurality of walls having a first transfer opening extending therethrough for communication with the compartment; a first gas permeable membrane disposed on the housing so as to cover at least a portion of the first transfer opening, the first gas permeable membrane having an inner side in direct communication with the compartment and an opposite outer side in direct communication with an environment external to the compartment; and A mixing element is movably disposed within the compartment of the housing.

2. The bioreactor of claim 1, wherein the first gas permeable membrane comprises a gas permeable silicone, dimethyl silicone, FEP or expanded polytetrafluoroethylene (ePTFE) sheet.

3. The bioreactor of claim 1, wherein no open pores are present in the first gas permeable membrane such that gas can only pass through the first gas permeable membrane at a molecular level by applying a diffusion gradient across the first gas permeable membrane.

4. The bioreactor of claim 1, wherein the gas permeable membrane has a flow rate of less than 75,000 mL / (m 2 *day)、100,000mL / (m 2 *day), 125,000mL / (m 2 *day) or 150,000mL / (m 2 *day) gas permeability.

5. The bioreactor of claim 1, wherein the first gas permeable membrane is liquid impermeable.

6. The bioreactor of claim 1, wherein each of the plurality of walls of the housing is gas and liquid impermeable.

7. The bioreactor of claim 1, wherein the housing is sufficiently rigid such that the housing does not flex when the compartment is filled with water.

8. The bioreactor of claim 1 , wherein the selected one of the plurality of walls has an inner surface communicating with the compartment and an opposite outer surface, and the first gas permeable membrane is mounted on the outer surface of the selected one of the plurality of walls.

9. The bioreactor according to claim 1, further comprising: said first gas permeable membrane having said inner side and said opposite outer side each extending to a surrounding peripheral edge; and A support frame is secured to the peripheral edge so as to surround the first gas permeable membrane, the support frame securing the first gas permeable membrane to the selected one of the plurality of walls.

10. The bioreactor of claim 9, wherein the support frame is secured to the selected one of the plurality of walls by welding or adhesive.

11. The bioreactor of claim 1 , wherein the mixing element comprises an impeller, blades or paddles rotatably disposed within the compartment.

12. The bioreactor of claim 1, further comprising a drive shaft having a first end and an opposing second end, at least a portion of the drive shaft being disposed within the compartment of the housing and having the mixing element disposed thereon.

13. The bioreactor of claim 12, wherein at least a portion of the drive shaft has a helical configuration.

14. The bioreactor of claim 12, wherein the drive shaft comprises: a first drive shaft portion, the first drive shaft portion having a helical configuration; and A second drive shaft portion having a helical configuration is provided, the second drive shaft portion being laterally spaced from and extending along the first drive shaft portion.

15. The bioreactor of claim 12, a drive motor mounted on the housing outside the compartment and coupled to the drive shaft.

16. The bioreactor of claim 1, further comprising a first sensor disposed at least partially within the compartment.

17. The bioreactor of claim 16, wherein the first sensor comprises a temperature sensor, a pH sensor, a DO sensor, or a CO2 sensor.

18. The bioreactor of claim 1, further comprising a port formed on the housing and communicating with the compartment, a gas filter coupled to the port.

19. The bioreactor of claim 1, wherein the plurality of walls of the housing are rigid.

20. The bioreactor of claim 1, wherein the plurality of walls comprises a top end wall, a bottom end wall, and a surrounding side wall extending between the top end wall and the bottom end wall.

21. The bioreactor of claim 20, further comprising: the first transfer opening, the first transfer opening extending through the surrounding sidewall; and A drive shaft protrudes from the top end wall into the compartment and toward the bottom end wall, the mixing element being arranged on the drive shaft.

22. The bioreactor according to claim 21 further comprises a stabilizing support member mounted on the inner surface of the bottom end wall so as to be connected to the compartment, the stabilizing support member having a top surface having a recess formed thereon, the terminal end of the drive shaft being received in the recess.

23. The bioreactor of claim 22, further comprising: the stabilizing support member, the stabilizing support member having a boundary surface defining the recess; and An annular lip seal projects radially inwardly from the boundary surface and forms a liquid-tight seal against the terminal end of the drive shaft.

24. The bioreactor of claim 22, further comprising: the stabilizing support having an interior surface at least partially defining a cavity; and A plurality of spaced apart gas openings extend between the top surface and the interior surface of the stabilizing support.

25. The bioreactor of claim 20, further comprising: a sprinkler mounted on the bottom end wall and communicating with the compartment; and A gas pipeline is coupled to the sprayer.

26. The bioreactor of claim 20, wherein the surrounding sidewall has a rectangular transverse cross-section.

27. The bioreactor of claim 20, wherein the surrounding side wall has a front wall having the first transfer opening extending therethrough, a rear wall opposite the front wall, and opposing side walls each extending between the top end wall and the bottom end wall.

28. A bioreactor according to claim 27, wherein the front wall has an external surface with an area, the first transfer opening extends through the front wall and has an area, the area of ​​the external surface includes the area of ​​the first transfer opening, and the area of ​​the first transfer opening is at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the area of ​​the external surface.

29. A bioreactor according to claim 27, wherein the front wall has a maximum height extending between the top end wall and the bottom end wall, and has a maximum width extending between the opposing side walls, and the maximum height is at least 1.2 times, 1.4 times, 1.6 times, 1.8 times, 2 times, 2.5 times, 3 times, 4 times or 5 times the maximum width.

30. The bioreactor of claim 29, wherein the maximum height of the front wall is at least 0.2 m, 0.3 m, 0.4 m, 0.6 m, 0.8 m, 1 m.

31. The bioreactor of claim 27, further comprising: a plurality of first mounting portions formed on an outer surface of the front wall; and A plurality of second mounting portions are formed on an outer surface of the rear wall, and the first mounting portion is configured to engage with the second mounting portions.

32. The bioreactor of claim 31, wherein at least the first mounting portion protrudes outwardly from the outer surface of the front wall, or the second mounting portion protrudes outwardly from the outer surface of the rear wall.

33. The bioreactor of claim 20, further comprising: a second transfer opening extending through the bottom end wall so as to communicate with the compartment; and A second gas permeable membrane is disposed on the housing so as to cover at least a portion of the second transfer opening.

34. The bioreactor of claim 1, further comprising a heater support, the heater support comprising: a body having a top surface with a recessed cavity therein; and one or more heating elements, the one or more heating elements being disposed within the body, Wherein at least a portion of the housing is removably received within the recess of the body.

35. The bioreactor of claim 1, wherein the first gas permeable membrane allows gas to pass therethrough only by passive gas exchange through diffusion.

36. The bioreactor of claim 1, wherein the first gas permeable membrane comprises a single continuous panel or sheet, rather than two or more panels or sheets that are overlapped and coupled together.

37. A bioreactor, comprising: a housing comprising a plurality of walls defining a compartment adapted to hold a liquid, a selected one of the plurality of walls having a transfer opening extending therethrough for communication with the compartment; a gas permeable membrane disposed on the housing so as to cover at least a portion of the transfer opening, the gas permeable membrane comprising a gas permeable silicone, dimethyl silicone, FEP, or expanded polytetrafluoroethylene (ePTFE) sheet; and A mixing element is movably disposed within the compartment of the housing.

38. A bioreactor, comprising: a housing comprising a plurality of walls defining a compartment adapted to hold a liquid, a selected one of the plurality of walls having a transfer opening extending therethrough for communication with the compartment; a gas permeable membrane disposed on the housing so as to cover at least a portion of the transfer opening, wherein no open pores are present in the gas permeable membrane such that gas can only pass through the gas permeable membrane at a molecular level by applying a diffusion gradient across the gas permeable membrane; and A mixing element is movably disposed within the compartment of the housing.

39. A bioreactor, comprising: a housing comprising a plurality of walls defining a compartment adapted to hold a liquid, a selected one of the plurality of walls having a transfer opening extending therethrough for communication with the compartment; a gas permeable membrane disposed on the housing so as to cover at least a portion of the transfer opening, wherein gas can only pass through the gas permeable membrane at a molecular level by applying a diffusion gradient across the gas permeable membrane; and A heater support, the heater support comprising: a body having a top surface with a recessed cavity therein; and one or more heating elements, the one or more heating elements being disposed within the body, Wherein at least a portion of the housing is removably received within the recess of the body.

40. A bioreactor, comprising: a housing including a top wall, a bottom wall, and a surrounding side wall extending between the top wall and the bottom wall, the housing defining a compartment; a first transfer opening extending through the side wall so as to communicate with the compartment; a first gas permeable membrane disposed on the housing so as to cover at least a portion of the transfer opening; a second transfer opening extending through the bottom wall so as to communicate with the compartment; and A second gas permeable membrane is disposed on the housing so as to cover at least a portion of the second transfer opening.

41. The bioreactor of claim 40, further comprising a mixing element movably disposed within the compartment of the housing.

42. A method for expanding cells in a first bioreactor, the first bioreactor comprising: a housing including a plurality of walls defining a compartment adapted to hold a liquid, the plurality of walls including a top end wall, a bottom end wall, and a surrounding side wall extending between the top end wall and the bottom end wall, the surrounding side wall having a front face, an opposed rear face, and opposed sides each extending between the top end wall and the bottom end wall, a first transfer opening extending through the front face for communicating with the compartment; and a gas permeable membrane disposed on the housing so as to cover at least a portion of the first transfer opening, the method comprising: placing the first bioreactor in a first orientation such that the first gas permeable membrane faces downward or sideways; expanding cells within the compartment of the housing for a first period of time while the first bioreactor is in the first orientation; rotating the first bioreactor to a second orientation such that the first gas permeable membrane faces sideways; and The cells within the compartment of the housing are expanded for a second period of time while the first bioreactor is in the second orientation.

43. The method of claim 42, wherein the first gas permeable membrane has a relative humidity of less than 75,000 mL / (m 2 *day)、100,000mL / (m 2 *day), 125,000mL / (m 2 *day) or 150,000mL / (m 2 *day) gas permeability.

44. The method of claim 42, wherein the first gas permeable membrane is liquid impermeable.

45. The method of claim 42, wherein the first gas permeable membrane comprises a gas permeable silicone, dimethyl silicone, FEP, or expanded polytetrafluoroethylene (ePTFE) sheet.

46. ​​The method of claim 42, wherein no open pores are present in the first gas permeable membrane and gas can only pass through the first gas permeable membrane at a molecular level by applying a diffusion gradient across the first gas permeable membrane.

47. The method of claim 42, wherein the first period of time comprises at least 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 120 hours.

48. The method of claim 42, wherein the housing of the first bioreactor is not repeatedly shaken or rotated during the first time period.

49. The method of claim 42, wherein a mixing element is movably disposed within the compartment of the housing, and a drive motor is coupled to the mixing element such that activation of the drive motor facilitates movement of the mixing element, wherein the drive motor is not activated to facilitate movement of the mixing element during the first time period.

50. The method of claim 49, further comprising activating the drive motor to facilitate movement of the mixing element during the second time period.

51. The method of claim 42, further comprising mounting a gas sparger on a bottom end wall of the housing and in communication with the compartment, wherein gas does not pass through the gas sparger during the first time period.

52. The method of claim 51, further comprising spraying gas through the gas sprayer during the second time period.

53. A method according to claim 42, wherein the compartment of the first bioreactor has a central longitudinal axis extending through the top end wall and the bottom end wall, and when in the first orientation, the central longitudinal axis is set at any angle between + / - 20°, 15°, 10° or 5° relative to the horizontal plane.

54. A method according to claim 42, wherein the compartment of the first bioreactor has a central longitudinal axis extending through the top end wall and the bottom end wall, and when in the second orientation, the central longitudinal axis is set at any angle between + / - 20°, 15°, 10° or 5° relative to the vertical plane.

55. The method of claim 42, further comprising placing the first bioreactor within an incubator such that the first bioreactor is disposed within the incubator during at least a portion of the first time period.

56. The method of claim 55, removing the first bioreactor from the incubator after the first time period such that the first bioreactor is disposed outside of the incubator during at least a portion of the second time period.

57. The method of claim 42, further comprising inserting a portion of the housing into a recess of a heater support after rotating the first bioreactor to the second orientation.

58. The method of claim 42, wherein the cell is a mammalian cell.

59. The method of claim 58, wherein the mammalian cell is a human cell.

60. The method of claim 58, wherein the cell is of a type selected from the group consisting of: a. Immune system cells, b. Stem cells, c. primary cells, and d. Immortalized cells.

61. The method of claim 60, wherein the immune system cell is of a type selected from the group consisting of: a. Natural killer cells, bT cells, cB cells, d. Antigen presenting cells, and e. Dendritic cells.

62. The method of claim 42, wherein the cells are expanded in culture.

63. The method of claim 62, wherein the culture medium is a serum-free medium.

64. The method of claim 62, wherein the culture medium comprises one or more chemokines or cytokines.

65. The method of claim 64, wherein the one or more chemokines or cytokines are one or more proteins selected from the group consisting of: (a) Interleukin-1α, (b) interleukin-2, (c) interleukin-4, (d) interleukin-1β, (e) interleukin-6, (f) interleukin-12, (g) interleukin-15, (h) interleukin-18, (i) interleukin-21, and (j) Transforming growth factor β1.

66. The method of claim 60, wherein the immune system cell is a T cell.

67. The method of claim 66, wherein the T cell is contacted with one or more agents that bind to one or more cellular receptors present on the T cell.

68. The method of claim 67, wherein the one or more agents comprise one or more antibodies that bind to one or more T cell surface receptors selected from the group consisting of: a.CD3, b.CD5, c.CD28, d.CD137, and e.CD278.

69. A method for introducing one or more materials into a mammalian cell, the method comprising contacting the mammalian cell with the material under conditions that allow the mammalian cell to uptake the one or more materials, wherein after the mammalian cell is contacted with the one or more materials, the cell is incubated in a bioreactor according to any one of claims 1 to 41.

70. The method of claim 69, wherein uptake of the one or more materials by the mammalian cells is mediated by transduction.

71. The method of claim 70, wherein the transduction method is electroporation.

72. The method of claim 71, wherein the mammalian cells are maintained in the bioreactor at less than 40 impeller RPM for at least 30 minutes after electroporation.

73. The method of claim 71, wherein the mammalian cells are electroporated in the bioreactor.

74. The method of claim 71, wherein the mammalian cells are transferred to the bioreactor after electroporation.

75. The method of claim 70, wherein the transduction method is viral transduction.

76. The method of claim 75, wherein the viral transduction is lentiviral transduction.

77. The method of claim 76, wherein the mammalian cells are contacted with the lentivirus in the bioreactor.

78. The method of claim 77, wherein the mammalian cells are maintained in the bioreactor at less than 40 impeller RPM for at least 30 minutes after contacting with the lentivirus.

79. The method of claim 69, wherein the one or more materials are selected from the group consisting of: a.Cas9 / guide RNA complex, b. a nucleic acid encoding a Cas9 protein, c. a nucleic acid encoding a guide RNA, and d. Nucleic acid encoding a chimeric antigen receptor.

80. A bioreactor, comprising: a housing including a bottom wall and at least one side wall upstanding from the bottom wall, the housing defining a compartment; a mixing element disposed within the compartment of the housing; a transfer opening extending through the at least one side wall; and A gas permeable membrane covers at least a portion of the transfer opening.

81. The bioreactor of claim 80, wherein the mixing element comprises a helical shaft or member.

82. The bioreactor of claim 81 , wherein the helical shaft or member is flexible.

83. The bioreactor of claim 81 , wherein the helical shaft or member is rigid.

84. A dual-mode bioreactor, the dual-mode bioreactor comprising: a housing including a bottom wall and at least one side wall upstanding from the bottom wall, the housing defining a compartment having a longitudinal axis extending therethrough; a mixing element disposed within the compartment; a transfer opening extending through the at least one side wall; and a gas permeable membrane covering at least a portion of the transfer opening; Wherein the bioreactor is configured to operate the mixing element when the housing and the longitudinal axis are in a vertical orientation.

85. The dual mode bioreactor of claim 84, further configured to operate the mixing element when the housing and longitudinal axis are in a horizontal orientation.

86. The dual mode bioreactor of claim 84, further configured to stop or prevent operation of the mixing element when the housing and longitudinal axis are in a horizontal orientation.

87. The dual mode bioreactor of claim 84, wherein the mixing element comprises a helical shaft or member.

88. The dual mode bioreactor of claim 87, wherein the helical shaft or member is flexible.

89. The dual mode bioreactor of claim 87, wherein the helical shaft or member is rigid.

90. A bioreactor, comprising: a support housing including a surrounding sidewall at least partially defining a chamber, an access opening extending through the sidewall to communicate with the chamber; a bag assembly disposed at least partially within a chamber of the support housing, the bag assembly comprising: a collapsible bag comprised of one or more sheets of polymeric film and defining a compartment, a transfer opening extending through a portion of the collapsible bag so as to communicate with the compartment; and a gas permeable membrane secured to the collapsible bag so as to at least partially cover the transfer opening, the gas permeable membrane being aligned with the access opening of the support shell; and A mixing element is movably disposed within the compartment of the collapsible bag.

91. The bioreactor of claim 90, further comprising a support structure spanning the inlet opening of the support housing and supporting the gas permeable membrane.

92. A bioreactor capsule, the bioreactor capsule comprising: The base of the cabin box; a bioreactor removably coupled to the pod base; a first pod module for conveying a first fluid into and out of the bioreactor and regulating a flow rate of the first fluid into and out of the bioreactor; a display including a user interface for sending operator inputs and receiving process parameter outputs associated with the bioreactor; and A controller including a memory operatively associated with the processor for controlling components associated with the bioreactor or components associated with the pod module.

93. The bioreactor cartridge of claim 92, wherein the bioreactor is a dual-mode bioreactor capable of culturing cells in the cell culture medium in a static mode in which the cell culture medium is not mixed, and in a dynamic mode in which the cell culture medium is mixed.

94. The bioreactor capsule of claim 92, wherein the bioreactor comprises a top end wall having a first top port and a bioreactor base having a first bottom port.

95. The bioreactor cartridge of claim 94, further comprising a gas overlap assembly coupled to the first top port and a sparger coupled to the first bottom port.

96. The bioreactor cartridge of claim 94, wherein the top end wall further comprises a second top port and a first sensor assembly coupled to the second top port for measuring a process parameter.

97. A bioreactor chamber according to claim 96, wherein the process parameters include the pressure within the bioreactor, the temperature of the cell culture medium, the foam content within the bioreactor, the glucose content of the cell culture medium, the pH of the cell culture medium, the dissolved oxygen content of the cell culture medium, the CO2 content of the cell culture medium or the cell density of the cell culture medium.

98. The bioreactor pod of claim 92, wherein the first pod module is detachable from the pod base.

99. The bioreactor cartridge of claim 92, wherein the first cartridge module is a first pump module for pumping the first fluid to the bioreactor, and the first fluid is a liquid containing cells.

100. The bioreactor cartridge of claim 99, wherein the first pump module comprises two pumps.

101. The bioreactor pod of claim 92, further comprising a second pod module for delivering a second fluid to the bioreactor and regulating a flow rate of the second fluid to the bioreactor.

102. The bioreactor pod of claim 101, wherein the second pod module is detachable from the pod base.

103. The bioreactor cartridge of claim 101, wherein the second cartridge module is a second pump module for pumping the second fluid to the bioreactor, and the second fluid is the cell culture medium.

104. The bioreactor cartridge of claim 103, wherein the second pump module comprises two pumps.

105. The bioreactor pod of claim 95, further comprising a third pod module for delivering a third fluid to the bioreactor and regulating a flow rate of the third fluid to the bioreactor.

106. The bioreactor pod of claim 105, wherein the third pod module is detachable from the pod base.

107. The bioreactor cartridge of claim 105, wherein the third cartridge module is a mass flow controller and the third fluid is oxygen.

108. The bioreactor pod of claim 105, wherein the third pod module is fluidly connected to the gas overlap assembly and the sparger to deliver oxygen into the bioreactor through the gas overlap assembly and the sparger.

109. The bioreactor capsule of claim 92, wherein the controller is housed within the bioreactor base.

110. The bioreactor pod of claim 94, wherein the pod base comprises a heating element for heating the bioreactor base.

111. The bioreactor capsule of claim 92, wherein the bioreactor comprises an impeller assembly comprising: Impeller shaft; three three-blade blades vertically spaced apart and coupled to the impeller shaft; and Impeller mounting hub.

112. The bioreactor cartridge of claim 111, wherein the impeller mounting hub comprises: a mounting shaft removably coupled to the impeller shaft; Install the wheel hub; and A first bearing engages the mounting shaft within the mounting hub.

113. The bioreactor cartridge of claim 112, wherein the impeller mounting assembly comprises two supports laterally spaced apart from the impeller shaft.

114. The bioreactor pod of claim 112, further comprising a motor comprising a motor shaft removably coupled to the mounting shaft.

115. The bioreactor pod of claim 114, further comprising a bracket coupling the display to the pod base.

116. The bioreactor cartridge of claim 115, wherein the bracket includes a motor mounting portion for mounting the motor when the motor is disengaged from the mounting shaft.

117. The bioreactor cartridge of claim 115, wherein the support comprises a scanner for scanning one or more bar codes associated with the cell culture medium, the bioreactor, the first module, the second module, the third module, the fourth module, the gas overlap assembly, the sprinkler, the first sensor assembly, and the motor.

118. A bioreactor cartridge according to claim 115, wherein the one or more bar codes indicate properties of the cell culture medium, the bioreactor, the first module, the second module, the third module, the fourth module, the gas overlap assembly, the sprinkler, the first sensor assembly, or the motor.

119. The bioreactor cabin according to claim 114 further comprises a fourth cabin module, the fourth cabin module comprising a power supply for providing power to the bioreactor cabin and an emergency stop device for stopping power supply to the first cabin module, the second cabin module, the third cabin module, the first sensor assembly and the motor.

120. The bioreactor pod of claim 119, wherein the fourth pod module is detachable from the pod base.

121. The bioreactor pod of claim 119, wherein the fourth pod module is housed within the pod base.

122. The bioreactor capsule of claim 96, wherein the first sensor assembly comprises: three foam sensors; Dip tube; and resistance temperature detectors.

123. A bioreactor chamber box according to claim 122, wherein the first sensor assembly further comprises three foam sensor sheaths each surrounding one of the three foam sensors and a temperature sensor sheath surrounding the resistance temperature detector, wherein the three foam sensor sheaths and the temperature sensor sheath each have an opening at one end to facilitate communication between the three foam sensors and the resistance temperature detector and the cell culture medium.

124. The bioreactor capsule of claim 93, wherein the bioreactor comprises a housing comprising a transfer opening and a diffusion gas permeable membrane covering at least a portion of the transfer opening.

125. The bioreactor cartridge of claim 124, wherein the diffusion gas permeable membrane allows gas to pass therethrough only by passive gas exchange via diffusion.

126. The bioreactor capsule of claim 125, wherein the diffusing gas permeable membrane comprises no pores.

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