Cell Culture Bioreactor with Rotational Mixing

Abstract

The cell culture bioreactor optionally has a hollow fiber membrane within the extracapillary space, used for growing cells in suspension. The membrane can include a liquid perfusion membrane and / or a gas transfer membrane. Liquid medium moves relative to the membrane within the extracapillary space. The bioreactor can be rotated around an axis of rotation that passes through the bioreactor in a repeating clockwise and counterclockwise pattern. The axis of rotation can be horizontal, pass through the center of the bioreactor, and / or perpendicular to the membrane. The rotation pattern can include five or more direction changes and / or decelerations and accelerations (optionally stopping and restarting) in one direction per minute. The bioreactor can be rotated less than 360 degrees in one direction, then less than 360 degrees in the opposite direction. The rotation of the bioreactor can reach speeds ranging from 1 to 25 rpm.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of and priority to U.S. Patent Application No. 63 / 421,012, filed October 31, 2022. U.S. Patent Application No. 63 / 421,012 is incorporated herein by reference.

[0002] Field This specification relates to a method for cell culture and a cell culture bioreactor. [Background technology]

[0003] The following is not an admission that what is stated below is common general knowledge or that it is recitable as prior art.

[0004] The term "cell culture" is sometimes used to refer to the culture of any cells, and sometimes specifically to the culture of eukaryotic organisms. In this specification, unless otherwise specified, cell culture includes the culture of any cells, including: a) eukaryotic organisms, such as animal cells, e.g., mammalian cells; b) non-eukaryotic organisms, such as bacteria or eukaryotic organisms, e.g., yeast, fungi, or protozoa (sometimes referred to as "microbial cultures"); and c) plant cells (sometimes referred to as "plant cell cultures" or "tissue plant cell cultures"). Furthermore, as used herein, cell culture, unless otherwise specified, includes growing cells for the purpose of obtaining the cells themselves, as well as growing cells for the purpose of obtaining products produced by the cells, e.g., genetic material, proteins, peptides, or enzymes. This is in contrast to growing cells primarily for the purpose of consuming pollutants, such as those found in wastewater treatment.

[0005] In some cell culture bioreactors, the nutrient medium flows through the lumen of the hollow fiber membrane, providing a perfusion culture mode, where nutrients diffuse through the pores of the membrane to cells growing outside the membrane, but within the bioreactor, optionally called the extracapillary space (ECS).

[0006] International Publication No. WO 2021 / 155469 A1, Cell Culture Bioreactor with Zone Control, describes a cell culture bioreactor with a membrane divided into multiple zones. The membrane includes a perfusion membrane or a gas transport membrane, or both, that transports liquid medium. The supply of one or more gases or liquid medium to selected zones can be controlled. The bioreactor can be used to grow cells in suspension. Mixing within the bioreactor's ECS is provided by a mixer, forced flow of liquid through the ECS, or movement of the bioreactor. Summary of the Invention

[0007] introduction The following introduction is not intended to limit or define the scope of the claims.

[0008] This specification describes a system and process for providing mixing in the extracapillary space (ECS) of a membrane bioreactor. The membrane bioreactor can be used to grow cells in suspension, for example, to provide the cells or a substance produced by the cells as a product. The membrane bioreactor includes a membrane within a plenum that defines the exterior of the extracapillary space of the bioreactor. The membrane may include a liquid perfusion membrane or a gas transport membrane, or both.

[0009] The mixing method involves moving the liquid medium in the extracapillary space of the bioreactor relative to the membrane. In some instances, the relative movement is provided by moving the bioreactor. Moving the bioreactor can include rotating the bioreactor clockwise and counterclockwise around an axis of rotation. The axis of rotation may pass through the bioreactor. In some instances, the axis of rotation may be horizontal, pass through the center or axis of the bioreactor (e.g., a cylindrical or longitudinal axis), and / or be perpendicular to the membrane. The bioreactor may have hollow fiber membranes extending in multiple directions perpendicular or oblique to the axis of rotation and / or the axis of the bioreactor. The bioreactor can be rotated in a pattern that includes five or more direction changes and / or decelerations and accelerations in one direction per minute, optionally with stops and restarts. The bioreactor can be rotated less than 360 degrees in one direction, then less than 360 degrees in the opposite direction. The rotation of the bioreactor can reach speeds ranging from 1 to 25 or 2 to 15 rpm or more. Transferring can include inverting and re-inverting the bioreactor.

[0010] This specification also describes a method of growing cells in a bioreactor, comprising moving a liquid medium in a cell growth area of ​​the bioreactor by moving the bioreactor in a repeating pattern, optionally having a cycle time of 12 seconds or less. This specification also describes a method of growing cells in a bioreactor, comprising moving a liquid medium in a cell growth area of ​​the bioreactor by applying power, optionally by moving the bioreactor, wherein the applied power exceeds the average power for a material portion, i.e., for 20% or more, 25% or more, or 33% or more, optionally up to 50% of the movement cycle.

[0011] In some examples, a bioreactor used in any of the methods described herein has an axis, e.g., a longitudinal axis, and hollow fiber membranes extending in multiple directions, oblique or perpendicular to the axis. The bioreactor can define a generally cylindrical plenum, and the axis is the cylinder axis. A portion of the plenum can be defined by one or more interior surfaces of the bioreactor in the shape of a segment of a circle, cylinder, or cone centered on the axis. Moving the bioreactor can include rotating the bioreactor about the axis. The hollow fiber membranes can include gas transport membranes. The bioreactor can have membranes with an outer diameter of 0.3 mm or more. The membranes can be arranged in layers with spaces between adjacent layers. The membranes can be stacked with a spacing of at least 0.2 mm. The membrane packing density can be 25% or less.

[0012] The system can include a bioreactor, a stand for supporting the bioreactor, and a power unit. The bioreactor has an axis, e.g., a longitudinal axis, and hollow fiber membranes extending in multiple directions perpendicular or oblique to the axis. The hollow fiber membranes can include gas transport membranes. The stand can support the bioreactor with the axis of the bioreactor generally horizontal. The power unit is adapted to rotate the bioreactor about the axis. The power unit can be, for example, a motor or actuator, e.g., an electric, hydraulic, or pneumatic actuator.

[0013] In some examples, the bioreactor provides a plenum for growing cells, the plenum having an inner surface in the shape of a segment of a circle, a cylinder, or a cone centered on an axis. The inner surface can be provided by a potting head that holds the ends of the hollow fiber membranes. The plenum can be generally cylindrical, and the axis can be the cylindrical axis.

[0014] The system can also have a controller programmed or otherwise configured to generate a movement pattern for the bioreactor, such as any of the movements described herein, via a power unit. The power unit can be connected to the controller. In some examples, the movement pattern includes one or more of the following: a) rotating the bioreactor clockwise and counterclockwise about its axis; b) moving the bioreactor in a repeating pattern having a cycle time of 12 seconds or less; c) rotating the bioreactor in a pattern that includes five or more changes of direction and / or decelerations and accelerations (optionally stopping and restarting) in one direction per minute; d) rotating the bioreactor less than 360 degrees in one direction and then less than 360 degrees in the opposite direction in a repeating pattern; e) rotating the bioreactor at a rotation speed ranging from 1 to 25 or 2 to 15 rpm; f) moving the bioreactor according to any of the movements described herein or similar movements adjusted for different size reactors; and g) the bioreactor being inverted and re-inverted.

[0015] In some examples, the bioreactor includes multiple elements, each element including a set of membranes potted separately from the membranes in the other elements. In some examples, the membranes have an outer diameter of 0.3 mm or greater, the membranes are arranged in layers with spaces between adjacent layers, the membranes are stacked and spaced at least 0.2 mm apart, the membrane packing density is 25% or less, or 20% or less, or 15% or less, or in the range of 8-12%, and / or the volume of the extracapillary space of the bioreactor is less than 1 mm of surface area of ​​the gas transport membrane. 2 1-20, 2-20, or 2-10 mm per 3 The range is. [Brief explanation of the drawings]

[0016] [Figure 1A] Figure 1B shows the outside of the second part of the mold. Figure 1B shows the inside of the second part of the mold of Figure 1A. [Figure 2A]Figure 2B shows the inside of the first part of the mold. Figure 2B shows the outside of the first part of the mold of Figure 18A. [Figure 3A.3B-3C] 1 shows isometric, top and end views of a membrane plate assembly. [Figure 4] The mold is shown assembled using the first portion of the mold in Figures 2A and 2B, the second portion of the mold in Figures 1A and 1B, and the membrane plate assembly in Figures 3A-C. [Figure 5A-B] 5 shows an isometric view and a vertical cross-sectional view of the assembled mold of FIG. 4. [Figure 6] 1 shows a horizontal cross section of the mold with potting material added. [Figure 7] 7 shows an element made from part of the mold assembly of FIG. 6 with some of the potting material, mold, and membrane cut away and a cap added. [Figure 8] 1 shows a reactor with a magnetic base attached to a motor for rotating the reactor. [Figure 9A-9B] 1 shows another device for rotating the composite reactor. [Figures 10A-10B] 1 shows a composite reactor with headers and manifolds connected to its elements. [Figure 11] 1 shows a cell culture system. [Figure 12] 12 illustrates an alternative gas system for use with the cell culture system of FIG. 11. [Figure 13] 12 shows additional parts of the cell culture system of FIG. 11. [Figure 14] The results of the experimental trials are shown. DETAILED DESCRIPTION OF THE INVENTION

[0017] The use or description of the invention will be further enabled below by the description of one or more examples or embodiments of the invention.

[0018] The figure shows examples of a bioreactor element 402, a reactor 450 fabricated from a single element 402, and a composite reactor 550 fabricated from multiple elements 402. These and similar bioreactors are described in International Publication No. WO2020 / 069607 A1, "Cell Culture Bioreactor," published April 9, 2020, and International Publication No. WO2021 / 155469 A1, "Cell Culture Bioreactor with Zone Control," published August 12, 2021, both of which are incorporated herein by reference. The words "reactor" and "bioreactor" are used interchangeably herein unless otherwise noted. For example, a reactor 450 having a single element 402 or a composite reactor 550 having multiple elements 550 can be generally referred to as a reactor or a bioreactor. One or more of the inventions described herein can be adapted to other bioreactors described in these publications or known in the art.

[0019] An element 402, optionally referred to as a module, contains membranes 102. In the example shown, the membranes 102 are hollow fiber membranes, although other types of membranes can be used. The membranes 102 can be divided into sets of membranes 102. In the example shown, the membranes 102 include membranes oriented in two directions. In other examples, the membranes 102 can be oriented in more or fewer directions. In the example shown, there are also two types of membranes: perfusion membranes 102a and gas transport membranes 102b. In other examples, the element can have, for example, only perfusion membranes 102a, only gas transport membranes 102b, a mixture of different perfusion membranes 102a, or a mixture of different gas transport membranes 102b. In the example shown, the perfusion membranes 102a are perpendicular to the gas transport membranes 102b. The membranes 102 can provide liquid medium perfusion or gas perfusion while retaining the cells within the ECS. Optionally, the pore size or skin of membrane 102 also retains selected media components or cell culture products within the ECS.

[0020] In the example shown, the composite reactor 550 has three elements 402. However, another composite reactor 550 may have more or less than three elements 402. The extracapillary spaces of the elements 402 within the composite reactor 550 are in fluid communication with one another and collectively form one continuous plenum inside the reactor 150. The number of elements 402 is optionally sufficient so that the height of the plenum (measured perpendicular to the membranes 102 in the example shown) is 50% or more, 100% or more, or 200% or more of the (average) length of the hollow fiber membranes 102a and / or 102b (measured between the inner surfaces of the potting material 428). Alternatively, multiple reactors 450 can be attached to one another and moved together, but have separate extracapillary spaces, although such an assembly is not considered a composite reactor 550. Multiple reactors 450, 550 are optionally connected in parallel, sharing a perfusion medium and gas transfer system. Parallel reactors can be used, for example, for autologous cell therapy manufacturing. Multiple reactors 450, 550 can also be used in series, e.g., to expand the cell population by first growing cells in a smaller reactor 450, 550 and then transferring the cells to a larger reactor 450, 550.

[0021] In the example shown, reactor 450 or composite reactor 550 also includes a top plate 436 and a base plate 438, which may be a magnetic base plate 444. Optionally, elements 402 can be adhesively sealed to each other or to the top plate 436 and base plates 438, 444. Sensors or fittings can be located on the top plates 436, 444, the base plate 438, or on both sides of element 402, or in multiple locations. In some examples, a piece of optical sensor foil is located inside the translucent wall of element 402. The sensor foil emits a signal when interrogated with a detector unit through the transparent wall of element 402. The detector unit can have, for example, a light source of one or more peak wavelengths, a camera (i.e., CMOS or CCD) chip, and optionally one or more optical filters. Optionally, the VisiSens TD™ modular mapping system from PreSens can be used as the detector unit. Optionally, different sensors are provided at different locations in the reactor 450 or complex reactor 550. Optionally, the reactor 450 or complex reactor 550 can also have a collection layer, allowing for removal of cells or cell products.

[0022] In some examples, cells in suspension within reactor 450 or composite reactor 550 receive nutrients through perfusion membrane 102a and oxygen through gas transfer membrane 102b. Optionally, soluble or dispersed cell waste products can be removed through perfusion membrane 102a. A first culture medium (alternatively referred to as ECS medium) is typically added to the extracapillary space of reactor 450, 550 during the setup phase, for example, through fitting 164. A second liquid culture medium (alternatively referred to as perfusion medium) is circulated through lumen 144 of perfusion membrane 102a. The second culture medium may be the same as or a different medium from the first culture medium. Either the first culture medium or the second culture medium may be a mixture of two or more culture media. The composition of either the first culture medium or the second culture medium may change during use of reactor 450, 550.

[0023] Optionally, carbon dioxide released by the cells is removed from the extracapillary space of the bioreactor 150 through the gas transport membrane 102b and in solution through the perfusion membrane 102a. The gas transport membrane 102b can have engineered pores, e.g., 30 angstroms or less or 40 angstroms or less in size, or it may be dense-walled. In other examples, the gas transport membrane 102b has larger pores but is sufficiently hydrophobic to prevent the pores from filling with water. Optionally, oxygen-enriched air is supplied intermittently or continuously through the gas transport membrane 102a. The oxygen concentration of the supplied air, the pressure of the supplied air, and / or the flow rate of the supplied air may be varied over time to change the amount of oxygen delivered to the extracapillary space of the reactor 450, 550. For example, the amount of delivered oxygen may be increased over time, with oxygen being delivered at a higher rate as the cells or cell population matures.

[0024] In some examples, the reactor 450, 450 is used to grow cells in suspension. Cells in suspension can move within the extracapillary space, typically because they are entrained in a moving liquid flow within the extracapillary space. Flow can be induced by moving the reactor 450, 550. The cells can be suspended singly or in aggregates, or attached to a carrier that is present in the suspension. The cells can be, for example, stem cells, CD34+ cells, HEK cells, CHO cells, RBCs, or any other cells mentioned herein, including eukaryotic, microbial, or plant cells.

[0025] Optionally, the outer diameter of the membrane 102 may be 0.3 mm or more, 0.5 mm or more, 0.7 mm or more, or 1.0 mm or more, up to, for example, 3 mm. A larger diameter membrane 102 can withstand greater mechanical stress than a smaller diameter membrane. A larger diameter membrane 102 also allows for more even distribution of nutrients, especially for long membranes, for example, 20 cm or more.

[0026] The ability to grow and harvest cells, e.g., maintain at least a portion of the cells in suspension, or remove temporarily arrested cells, is also enhanced by having controlled spacing and / or low packing density between the membranes 102. The packing density of the membranes 102 (measured as the sum of the cross-sectional areas of the membranes divided by the cross-sectional area of ​​the potting material) may be 25% or less, 20% or less, or 15% or less. In addition, gaps may be provided between a set of membranes 102 and / or between a set of membranes 102 and the interior surface of the reactor 450, 550, as shown, for example, in FIG. 1 . Optionally, the membranes 102 are arranged in a regular pattern with controlled spacing between adjacent membranes 102. In the example shown, the membranes 102 are arranged in a stack of layers, optionally forming a linear array. Within a layer, adjacent membranes 102 are separated from each other by gaps (measured between the outer surfaces of adjacent membranes), e.g., 0.2 mm or more, 0.5 mm or more, 0.7 mm or more, or 1.0 mm or more. The layers of membrane 102 are separated from one another by gaps (measured between planes defining the facing surfaces of adjacent layers), e.g., 0.2 mm or more, 0.5 mm or more, 0.7 mm or more, or 1.0 mm or more. The gap between adjacent layers of membrane 102, if any, may also be at least as large as the diameter of a vertical membrane. The spacing between membranes 102 within a layer and / or between layers is optionally different for perfusion membranes 102b than for gas transport membranes 102a. The spacing between membranes 102 or between layers, the potting density, and the arrangement of membranes 102 into layers or arrays are preferably determined and / or measured within the potting material. The defined spacing is optionally maintained along the length of the membranes 102, for example, by tension within the membranes 102 or by weaving diagonal or orthogonal membranes 102 together.

[0027] The diameter of the element 402, measured between the inner surfaces of the potting material 428, may be in the range of, for example, 5 cm or more, 10 cm or more, 15 cm or more, or 20 cm or more. The diameter may be, for example, 40 cm or less, or 30 cm or less. In the example shown, the inner surface of the potting material defines a circle centered on the axis used for spin casting, which may be the axis 165 of the spinning or reactor 450, 550, as described further below. Depending on the orientation of the element 402 when the potting material is cast, the inner surface of the potting material may define a segment of a cylinder or cone, preferably a gently tapered cone. In the case of a conical element 402, the diameter of the element may be considered the average diameter. In examples where the element 402 does not have a segment defining a circular inner cross-section, one or more dimensions between opposing inner surfaces of the element 140 may be within these ranges. Longer membranes 102 can be strengthened if necessary by increasing their diameter, using membranes 102 of multifilament yarn, weaving orthogonal membranes 102 together, or using braided or TIPS membranes 102.

[0028] Optionally, element 402 can have additional partitions between membranes 102, forming more distinct sets of membranes 102. For example, the element 402 shown has four sets of membranes 102: two sets of perfusion membranes 102a and two sets of gas transfer membranes 102b. Other examples can have more or fewer sets of membranes 102, and there may be a different number of sets for perfusion membranes than for gas transfer membranes. Creating more sets can help maintain at least some cells in suspension, as the diameter of element 402 increases. In some examples, element 402 has most of its membranes 102, i.e., more than 50%, more than 80%, or all, in sets that are 10 cm or less, or 5 cm or less in width.

[0029] The element 402 can be used with the axis 165 perpendicular to the membrane 102 in a generally horizontal orientation, as shown, for example, in FIGS. 8, 9A, and 9B. The height of the reactor 450, 550, measured along the axis 165 in the ECS (i.e., horizontally in FIGS. 8, 9A, and 9B), can be greater than the inner diameter of the element 402. Optionally, the height of the reactor 450, 550 can be two or more, five or more, or ten or more times the inner diameter of the element 402. Optionally, one or more collection layers can be added between the elements 402. The extracapillary space of the bioreactor, measured as the internal volume of the bioreactor, excluding the volume occupied by the membrane 102, can be 0.01 L or more, 0.1 L or more, 1 L or more, 10 L or more, or 50 L or more. The extracapillary space of the bioreactor can be 1000 L or less, or 100 L or less, 20 L or less, or 10 L or less.

[0030] An exemplary bioreactor and method for manufacturing the same are described below. Figures 5A and 5B show a set of membranes 102 within a mold 400. The mold 400 can be used, for example, to manufacture the element 402 shown in Figure 7. In a casting process, a liquid potting material 428, such as an epoxy or polyurethane resin, is poured into the mold 400 and then hardens to form the solid shown in Figure 7. In the example shown, the mold 400 is used for spin casting. The mold 400 is spun as the liquid potting material 428 is added to the mold, forcing the potting material 428 to the outside of the mold 400. The mold 400 continues to spin until the liquid potting material 428 solidifies. Alternatively, static potting can be used. In this case, the portion of the mold 400 defining the potting cavity 416 is oriented at the bottom of the mold 400, and the liquid potting material 428 is poured into it and then hardened. The mold 400 is then rotated, another potting cavity 416 is placed in the bottom of the mold, and more potting material 428 is added. The static potting process is repeated until potting material 428 has been poured into each potting cavity 416. Spin casting is preferred, and generally cylindrical elements 402 can be produced. However, static potting can also be used, and optionally polygonal elements 402 can be produced, for example, square or octagonal elements 402 if angled panels 408 are present in the mold 400.

[0031] The potting material 428 is surrounded by a portion of the mold 400, except for the outer surface of the potting material 428, and the ends of the membrane 102 are open, exposing the lumen of the membrane 102. The inner surface of the potting material is within the mold 400, but is not in direct contact with the surface of the mold 400.

[0032] In the example shown in FIG. 5A, mold 400 is made of a transparent plastic, such as polycarbonate. A portion of mold 400 forms panel 408. Panel 408 allows for viewing into the extracapillary space or for determining properties of the first medium in the ECS using light-based analytical methods. Optionally, panel 408 allows for measurements to be taken from one or more light-activated sensor foils positioned inside panel 408 in contact with the first medium in the extracapillary space. Alternatively, mold 400 may be made of an opaque material, and other forms of sensors may be positioned on or through panel 408. Optionally, mold 400 can be reconfigured to create a larger area for potting material 428 and to eliminate or reduce the size of panel 408.

[0033] 5A and 5B, mold 400 has a first portion 404 and a second portion 406. First portion 404 and second portion 406 are assembled together, optionally with an adhesive, after membrane 102 is inserted between them. Once mold 400 is assembled, a potting cavity 416 is formed, where potting material 428 will be added (e.g., as shown in FIG. 6). During the spin-casting process, liquid potting material 428 flows into potting cavity 416 of mold 400 through one or more resin ports 412.

[0034] The element 402 has two openings 410 on opposite sides of the second element 402. Optionally, one or both of the openings 410 include additional features, such as a raised ring in the example shown. Alternatively, the openings 410 may be simple openings in the mold 400. Optionally, the outer diameter of one of the openings 410 is generally the same as the inner diameter of the other opening 410. Multiple elements 402 can be stacked together by inserting a smaller opening 410 of a second element 402 into a larger opening of another second element 402. Optionally, the openings 410 of two or more second elements 402 are joined by an adhesive or solvent-bonded together. Alternatively, the openings 410 may be threaded so that two or more second elements 402 can be screwed together, or two openings 410 can be press-fit together.

[0035] 1A and 1B show the second portion 406 of the mold 400. FIG. 1A shows primarily the outer surface of the second portion 406. FIG. 1B shows primarily the inner surface of the second portion 406. The opening 410 can have one or more registration areas 414. In the example shown, there are four registration areas 414 evenly spaced around the opening 410. Each registration area 414 is a flat spot on the otherwise circular opening 410. In combination with corresponding registration areas on the first portion 404 of the mold 400, the registration areas align the potting cavities 426 in the stack of second elements 402 with each other.

[0036] 2A and 2B show the first portion 404 of the mold 400. FIG. 2A primarily shows the inner surface of the second portion 406. FIG. 2B primarily shows the outer surface of the second portion 406. The opening 410 can have one or more registration areas 414. In the example shown, there are four registration areas 414 evenly spaced around the opening 410. Each registration area 414 is a flat spot on the otherwise circular opening 410. As described above, in combination with corresponding registration areas 414 on the second portion 406 of the mold 400, the registration areas 414 align the potting cavities 416 in the stack of second elements 402 with one another.

[0037] In the example shown, the mold 400 has four potting chambers 416, to which potting material 428 is applied. Optionally, during the potting process, the potting material 428 (e.g., as shown in FIG. 6 ) does not extend radially inward beyond the potting chambers 416. Thus, the potting material does not flow through the inside of the mold 400 between the potting chambers 416, and the inner surface of the potting material 428 may form a segment of a cylinder or cone. In other examples, the potting material 428 may extend radially inward beyond the potting chambers and flow between them, thus forming a continuous cylinder or cone. Each potting chamber 416 has one or more resin ports 412. During potting, the potting material 428 flows into the potting chambers 416 from a reservoir outside the mold 400 through a tube connected to the resin port 412. The potting material can be flowed by a pump or by centrifugal force created by spinning the reservoir with the mold 400 .

[0038] As shown in FIG. 2A , each potting chamber 416 also has one or more ribs 418. In the example shown, there is one rib 418 on each side of each potting chamber 416. The ribs 418 engage notches 420 in plates 422, shown in FIGS. 3A, 3B, and 3C. The membrane 102 is attached to the plates 422, for example, by adhesive or welding, to form a membrane plate assembly 426. The edges of the membrane 102 are typically closed before or after attachment to the plates 422. The ribs 418 position the plates 422 when they are inserted into the potting chamber 416. Alternatively, other features of the mold 400 and / or membrane plate assembly 416 can be used to hold the membrane plate assembly 416 in a selected location within the mold 400. The length of the membrane 102 can be selected for the configuration of the mold 400 and plates 422 so that the membrane 102 is taut when placed within the mold. Alternatively, the length of the membranes 102 can be selected relative to the configuration of the mold 400 and plates 422 so that the membranes 102 have some slack when placed within the mold 400. The membranes 102 can be divided into one or more sets of membranes 102 on the plates 424. Optionally, the membranes 102 in a set are evenly spaced from one another. Uniform spacing of the membranes 102 combined with taut membranes 102 promotes controlled, uniform spacing of the membranes 102 within the extracapillary space. Alternatively, loose membranes 102 may move more in response to mixing, which can inhibit cell attachment or aid cell collection for some cell types. The thickness of the plates 422, including the optional spacing blocks 424, can be varied to control the distance between the membranes 102 attached to one plate 422 and the membranes 102 attached to another plate 422.

[0039] The membrane plate assembly 426 can be customized, for example, by having one or more of a selected membrane 102 type or size, a selected spacing between membranes 102 in the membrane plate assembly 426, a selected spacing between membrane plate assemblies 126, a selected taut or loose mounting of the membranes 102, or a selected treatment of the membranes 102, for example, to make them resistant to protein fouling or environmentally (i.e., thermally) compliant. By modifying the membrane plate assembly 426, elements 402, reactors 450, or composite reactors 550 suitable for use in growing various cells or cell products can be generated. In some examples, the number of perfusion membranes 102a relative to the number of gas transport membranes 102b is varied, and only perfusion membranes 102a or only gas transport membranes 102b are provided in the element 402, reactor 450, or composite reactor 550.

[0040] FIG. 4 shows the mold 400 being assembled. A membrane plate assembly 426 is inserted into the first portion 404 of the mold 400. While two membrane plate assemblies 426 are shown in FIG. 4, the mold 400 can include multiple membrane plate assemblies 426, e.g., 5 to 1,000, or 10 to 100. In the example shown, there are perfusion membranes 102a and gas transport membranes 102b. Optionally, the mold 400 can be loaded with only perfusion membranes 102a or only gas transport membranes 102b. In the example shown, alternating membrane plate assemblies 426 are oriented orthogonally to one another. In other examples, the membrane plate assemblies 426 may form different patterns, e.g., two or three membrane plate assemblies 426 per direction, with one membrane plate assembly 426 per orthogonal to the other. In other examples, all of the membrane plate assemblies 426 in the mold 400 may be oriented in the same direction. In other examples, the membranes 102 of one membrane plate assembly 426 may be woven with the membranes 102 of an orthogonal membrane plate assembly 426. The membranes 102 of the membrane plate assembly 426 may be divided into the two sets shown, or into more than two sets. Optionally, the membranes 102 of the membrane plate assembly 426 may be uniformly spaced across substantially the entire width of the single set of membrane plate assembly 426.

[0041] 5A and 5B (described above) show the mold 400 assembled and ready for spin casing. FIG. 6 shows a cross section of the mold 400 after spin casting. Potting material 428 was added to each of the potting chambers 416, surrounding the edges of the membrane 102. Upon curing, the potting material 428 seals the outer surface of the membrane 102. Optionally, the potting material 428 also surrounds the plate 422. In the example shown, the inner surface of the potting material 428 is pulled away from the panel 408, i.e., radially displaced. Optionally, by adding more potting material 428 during spin casting, the inner surface of the potting material 428 is brought closer to or substantially flush with the edge of the panel 408. The panel 408 may be flat rather than curved as shown, so that the potting material 428 may intrude into the edges of the panel 408 and not flow all the way through the panel 408. Alternatively, some of the potting material 428 may overflow the panel 408 from one potting chamber 416 to another. The potting material 428 may be transparent, for example, a clear epoxy. In this case, the potting material 428 may cover the panel 408 and still allow light to pass through the panel 408.

[0042] In the example shown, the membrane 102 insets from the sidewall of the potting chamber 416. Alternatively, the membrane 102 may be positioned closer to the sidewall of the potting chamber 416. The size of the panels 408 (either their absolute size or their size relative to the size of the mold 400) may vary, or the panels 408 may be eliminated. The example shown is for a relatively small mold 400 having an outer diameter of approximately 10-15 cm. For example, in a larger mold 400 having an outer diameter of up to 30 cm or more, or up to 60 cm or more, panels 408 of essentially the same absolute size may be used, although the panels 408 would be relatively smaller in the larger mold 400. Thus, the portion of the volume of the extracapillary space not traversed by the membrane 102 can be reduced (or increased). However, it is not necessary or desirable to have more of the extracapillary space traversed by the membrane 102.

[0043] Having multiple potting chambers 416 divides the amount of potting material 428 into smaller units, which helps manage the heat generated as the potting material 428 cures and also promotes not coating the panel 408 with the potting material 428. Optionally, the mold 400 can be reconfigured to provide one continuous potting chamber 416, particularly if the panel 408 is not needed, for example, for a sensor or to enhance mixing.

[0044] FIG. 7 shows element 402. Starting with the potted mold 400 shown in FIG. 6, the potting material 428 and potting chamber 416 are cut to create a cut surface 432. The portion of the potting material 428 beyond the cut surface 432 and the edges of the membranes 102 are removed. Optionally, the cut surface 432 may be the inside of a plate 422, which may also be removed. The lumen of the membrane 102 is open at the cut surface 432. The cap 430 is sealed to the remaining components of the potting chamber 416, for example, with an adhesive or solvent. The edges of the membranes 102 are fluidly coupled to the inside of the cap 430. Cap ports 434 allow fluid to be added to or withdrawn from the cap 430, which in turn allows fluid to be added to or withdrawn from a set of membranes 102. In the example of FIG. 7, each cap 430 has a single cap port 434. Optionally, the cap 430 can have two or more cap ports 434 .

[0045] FIG. 8 shows a side view of the elements 402 used in reactor 450. A top plate 436 closes an upper opening 410 on the top surface of reactor 450. A magnetic base plate 444 closes a lower opening 410 on the bottom surface of reactor 450. Although only one of elements 402 is shown in FIG. 8, multiple elements 402, e.g., 2 to 100, or 2 to 10 elements 402, can be assembled together into a composite reactor 550, as described further below in connection with FIGS. 9A and 9B. For example, the opening 410 of one element 402 can be attached to the opening of another element 402 to create a stack of elements 402.

[0046] Referring to Figure 8, the top plate 436 can have one or more fittings 164, which provide access to the extracapillary space. The fittings 164 can be used to add a first medium or specific substances, such as growth factors or nutrients, to the extracapillary space. Alternatively, the fittings 164 can be used to remove substances from the extracapillary space. Optionally, the fittings 164 can be used to connect a sampler to the extracapillary space.

[0047] The top plate 436 can have one or more adapters 442. In one example, the adapter 442 is used to mount a sensor body connected to a fiber optic cable into a hole in the top plate 436. The fiber optic cable is used to read a sensor dot mounted inside the sensor body within the adapter 442. The sensor dot is manufactured, for example, by PreSens and can be used to measure pH, dissolved oxygen concentration, dissolved carbon dioxide concentration, or other aspects of the extracapillary space. Alternatively, the adapter 442 can be used to support another type of probe or sensor.

[0048] FIG. 8 shows a reactor 450 having a magnetic base 444 in place of the base plate 438 of FIGS. 10A and 10B. The magnetic base 444 has a ferromagnetic insert (not visible) that is held in place in the magnetic base 444. The ferromagnetic insert allows a second reactor 450 to be coupled to a magnet 440 outside of the reactor 450. The magnet 440 is attached to a motor 446, such as a stepper motor. The motor 446 is supported on a stand 448. The reactor 450 is thereby suspended from the stand 448. Alternatively, the motor 446 may be attached to the reactor 450 without a magnet or may be mechanically coupled (i.e., by a gear or drive belt). The reactor 450 may also be supported directly on the stand 448 (i.e., without relying on the motor 446 for support) or on a separate stand. The reactor 450 is rotated by the motor 446. The motor 446 can be activated to move the reactor 450, for example, to rotate or rock the second reactor 450, or to periodically invert the second reactor 450. In one example, the motor 446 rotates the reactor 450 0.1-5 revolutions in one direction (i.e., clockwise), then rotates the reactor 450 0.1-5 revolutions in the other direction (i.e., counterclockwise), and the pattern is repeated. When the reactor 450 is mixed by rotating or otherwise moving it, the potting material 428 can optionally be provided such that the inner surface of the potting material 428 is radially outwardly offset from the inner surface of the panel 408. In this manner, the panel 408 protrudes into the extracapillary space, forming vanes or ridges that can enhance the mixing caused by moving the reactor 450. The power terminals O, P, Q and R of the motor 446 are connected to a motor controller 640 (see FIG. 13 ), which is coupled to a power supply 642 and a computer 630 .Computer 630 can be programmed via motor control device 640 to operate motor 446 to rotate according to a predetermined pattern, such as a pattern to generate a mixture as described herein, or a mixing pattern that varies according to readings from one or more sensors, or to facilitate an action such as filling the ECS of reactor 450 or harvesting cells as needed.

[0049] Optionally, reactor 450 can have additional potting chambers 416, for example, six or eight potting chambers 416. In other examples, reactor 450 can have between two and twenty potting chambers 416. In some examples, the potting chambers 416 are distributed radially around reactor 450. In other examples, one or more sides of reactor 450 have no potting chambers or two or more potting chambers 416. Optionally, reactor 450 can have two different types of perfusion membranes 102a or two different types of gas transport membranes 102b. The different types of membranes may differ, for example, in pore size, material, or surface treatment.

[0050] 9A, 9B, 10A, and 10B show a composite reactor 550. The composite reactor 550 shown has three elements 402 stacked together. Alternatively, different numbers of elements 402, e.g., 2 to 20, may be stacked together. The elements 402 may all be the same size and configuration, or may have two or more different sizes or configurations. An opening 410 on the bottom surface of a first element 402 fits into an opening 410 on the top surface of another element 402 adjacent to the first element 402. The openings 410 of two different elements 402 are optionally glued together to enhance the seal between the openings 410. A base plate 438 or magnetic base plate 444 is connected to the openings 410 of the elements 402 on the bottom surface of the composite reactor 550. The extracapillary spaces of the second elements 402 are fluidly connected to each other through the openings 410, forming one larger extracapillary space for the entire composite reactor 550. However, the membrane 102 of each second element 402 is individually accessible through a cap port 434 on the cap 430 of the individual second element 402 .

[0051] The composite reactor 550 can have a collection layer 300 as well as one or more elements 402. The collection layer is similar to element 402 but has an exclusion membrane. The collection layer can be used to withdraw a portion of the first medium along with the product, while selectively excluding at least some of the producing cells remaining in the remainder of the first medium. The product can be, for example, enucleated red blood cells, viruses, proteins, or another cellular product. The exclusion membrane can have pores with a diameter of about 5 microns to retain nucleated red blood cell precursor cells or smaller diameters to selectively collect viruses or proteins.

[0052] Alternatively, the collection layer can have a port connected directly to the ECS (i.e., not through the membrane) and can be used to withdraw the cells for transfer to another reactor or for harvesting the cells as product.

[0053] FIG. 9A shows a composite reactor 550. The composite reactor 550 has a disk 512 inserted between a pair of second elements 402. Alternatively, or in addition, the disk 512 may be provided at the end of the composite reactor 550. The disk 512 rests on a pair of rollers 514. The rollers 514 provide another type of stand for holding the composite reactor 550. The rollers 514, disk 512, or composite reactor 550 can be driven by a motor, directly, or by a mechanical coupling, such as a gear or drive belt (motor and mechanical coupling not shown), to rotate the composite reactor 550 and provide mixing in the extracapillary space. Tubing for conveying fluids to or from the second element 402 can pass through holes or notches (not shown) in the disk 512. The disk 512 is annular and surrounds but does not block the opening 410, so the composite reactor 550 has one continuous ECS. Motor 446 (not shown) can be connected to roller 514 or directly to compound reactor 550. Motor 446 can be connected to computer 630 and can be operated as described for motor 446 in FIG.

[0054] FIG. 9B shows another composite reactor 550. This composite reactor 550 is mounted on both ends on a stand 448. One end of the stand 448 has a motor 446 that rotates a magnet 440. The magnet 440 is connected to a magnetic base 444 attached to one end of the composite reactor 550. The other end of the composite reactor 550 has a pinned top plate 516 supported on a bushing at the other end of the stand 448. Alternatively, the motor 446 can be attached to or mechanically coupled (i.e., by gears or a drive belt) to the reactor 450, 550, without a magnet. The reactor 450 can also be supported directly on the stand 448 (i.e., not relying on the motor 446 for support) or on a separate stand. The motor 446 can be used to rotate the composite reactor 550, providing mixing within the extracapillary space. The motor 446 can be connected to a computer 630 and operated as described for the motor 446 in FIG. 8.

[0055] Optionally, another form of power device can be used instead of a motor to move the reactor 450, 550. For example, the reactor 450, 550 can be moved by an actuator, such as an electric, pneumatic, or hydraulic actuator.

[0056] 8, 9A, and 9B show axis 165. Axis 165 is the axis of rotation. In the example shown, axis 165 is also the axis of reactor 450, 550. Axis 165 can be considered the longitudinal axis of reactor 450, 550 because the interior surface of the reactor's material, e.g., potting material 428, is symmetrical through several planes that include axis 165 and / or the cross-sectional shape of reactor 450, 550 varies less along axis 165 than the cross-sectional shape varies along other lines drawn through reactor 450, 550. In the example shown, axis 165 also passes through the center of top plate 436 and the center of base plate 438 or magnetic base 444. The interior surface of potting material 428 can define a segment of a circle, cylinder, or cone centered on axis 165. Although the panel 408 does not form a continuous circle, cylinder, or cone with the inner surface of the potting material 428, and although the inner surface of the potting material 428 may define a segment of a cone rather than a cylinder, the reactors 450, 550 can still be considered to define a plenum that is generally cylindrical with the axis 165 as the cylindrical axis. The membranes 102a, 102b can extend in multiple directions oblique to the axis 165. In the example shown, the membranes 102a, 102b extend in two directions that are both generally perpendicular to the axis 165.

[0057] FIG. 10A shows a composite reactor 550 having a liquid perfusion manifold 530. The liquid perfusion manifold 530 connects a medium supply point AA to a set of cap ports 434. In the example shown, these cap ports 434 are fluidly coupled to the upstream ends of the perfusion membranes 102a (not visible in FIG. 10A ) in the composite reactor 550. Optionally, a branch of the liquid perfusion manifold 530 has a control valve 538, which adjusts or stops the flow of medium to selected second elements 402. The composite reactor 550 also has a liquid perfusion header 532. The liquid perfusion header 532 connects the set of cap ports 434 to a medium collection point BB. In the example shown, these cap ports 434 are fluidly coupled to the downstream ends of the perfusion membranes 102a (not visible in FIG. 10A ) in the composite reactor 550. Optionally, a branch of the perfusion header 532 has a control valve 538, which adjusts or stops the flow of medium to selected elements 402. The perfusion manifold 530, perfusion header 532, and control valve 538 adjust the flow of medium to one set of elements 402 to all of the elements 402, or to a second set of elements 402. In some instances, having a single control valve 538 either upstream or downstream of an element 402 is sufficient. However, having control valves 538 both upstream and downstream of a compound reactor can provide additional control options. For example, choosing to adjust medium flow between the upstream control valve 538 and the downstream control valve 538 can affect the pressure inside the second element 402. In another example, one set of control valves 538 (either upstream or downstream of the composite reactor 550) can be linked together (e.g., mechanically, electrically, or in a control algorithm) to provide simultaneous regulation of medium flow to all of the secondary elements 402, while other sets of control valves 538 are individually controlled to regulate medium flow to one secondary element 402 relative to another secondary element 402.In the example shown, the liquid perfusion manifold 530 and liquid perfusion header 532 have three branches, but in other examples, the liquid perfusion manifold 530 and liquid perfusion header 532 can have a different number of branches corresponding to a different number of second elements 402 in the composite reactor 550. In other examples, the second medium flows sequentially through the elements 402 of the composite reactor 550. In this case, medium supply point AA is connected to the inlet cap port 434 of the first element 402. The outlet cap port 434 of the upstream element 402 is attached to the inlet cap port 434 of the downstream element 402. The outlet cap port 434 of the last element 402 is connected to the medium collection point BB.

[0058] FIG. 10B shows a composite reactor 550 having a gas perfusion manifold 534 connecting a gas feed point CC to a set of cap ports 434. In the example shown, these cap ports 434 are fluidly coupled to the upstream ends of gas transport membranes 102b in the composite reactor 550 (not visible in FIG. 32B ). Optionally, branches of the gas perfusion manifold 533 have control valves 538 that regulate or stop the flow of gas to selected secondary components 402. The composite reactor 550 also has a gas perfusion header 536 connected to a set of cap ports 434 to a gas recovery point DD. In the example shown, these cap ports 434 are fluidly coupled to the downstream ends of gas transport membranes 102b in the composite reactor 550 (not visible in FIG. 32B ). Optionally, branches of the gas perfusion header 536 have control valves 538 that regulate or stop the flow of medium to selected secondary components 402. The gas perfusion manifold 534, gas perfusion header 536, and control valves 538 regulate the flow of gas to all elements 402 or to one set of elements 402 relative to another set of elements 402. As described above, liquid medium, control valves 538 can be provided either upstream or downstream of the second elements 402, or both upstream and downstream. The selection between the upstream control valve 538 and the downstream control valve 538 for regulating gas flow can affect the pressure inside the second elements 402. Optionally, one set of control valves 538 (either upstream or downstream of the composite reactor 550) can be linked together (e.g., mechanically, electrically, or in a control algorithm) to provide simultaneous regulation of gas flow to all of the elements 402, while the other set of control valves 538 are individually controlled to provide regulation of gas flow to one element 402 relative to another element 402. In the example shown, the gas perfusion manifold 534 and the gas perfusion header 536 have three branches, but in other examples, they can have a different number of branches corresponding to a different number of second elements 402 in the composite reactor 550. In other examples, the gas flows sequentially through the elements 402 of the composite reactor 550.In this case, gas feed point CC is connected to the inlet cap port 434 of the first element 402. The outlet cap port 434 of the upstream element 402 is attached to the inlet cap port 434 of the downstream element 402. The outlet cap port 434 of the last element 402 is connected to the gas collection point DD.

[0059] 11 shows a cell culture system 600 including a reactor 450. Optionally, a composite reactor 550, optionally equipped with a liquid perfusion manifold 530, a liquid perfusion header 532, a gas perfusion manifold 534, and a gas perfusion header 536, can be used in place of reactor 450. The extracapillary space of reactor 450 can be filled with a first medium through fitting 164, while gas is released from another fitting 164, for example, through a valve (i.e., a pinch valve or a tubing clamp) and gas vent 612 (not shown). The extracapillary space can also be inoculated with cells to be grown in reactor 450 through fitting 164, while gas is released from another fitting 164, for example, through a valve (i.e., a pinch valve or a tubing clamp) and gas vent 612 (not shown). In the example shown, the same two fittings 164 are used to fill the reactor 450 with the inoculum and first medium, although additional dedicated fittings 164 can be provided for each task. After the extracapillary space is filled and inoculated, the fittings 164 are closed, for example, using a tube sealer or sterile disconnector, or used for other purposes. In some examples, the gas vent 612 includes a membrane with, for example, 0.22 micron pores, which allows gas to pass through but prevents bacterial contamination of the second reactor 450. In some examples, the gas vent membrane is hydrophobic and retains liquid. Optionally, the fitting 164, or an array of fittings 164, can be periodically or continuously connected to a sampler (not shown). The sampler can be used to withdraw a sample of the first medium, including cells and compounds dissolved and / or suspended in the first medium, from the extracapillary space. Optionally, one or more compounds, in addition to any compounds transferred to the ECS through perfusion membrane 102a, can be added to the extracapillary space through fitting 164. For example, growth factors can be added to the extracapillary space, e.g., to compensate for the decay or consumption of growth factors originally present in the first medium.

[0060] Reactor 450 has one or more sensors L, M, N in communication with the ECS through top plate 436. In the example shown, sensors L, M, N sense the pH, dissolved oxygen concentration, and dissolved carbon dioxide concentration of a first medium in the extracapillary space. Optionally, each sensor L, M, N is mounted to an adapter 442 over a hole in top plate 436. Sensors L, M, N have a probe body sealed to adapter 442. The probe body has a sensor dot in fluid communication with the first medium in the ECS. Adapter 442 also holds fiber optic cable 628 (shown in FIG. 13) in a position suitable for probing the sensor dot. Referring to FIG. 13, fiber optic cable 628 is connected to fiber optic meter 634. Fiber optic meter 634 is connected to computer 630, optionally via USB hub 632. The computer 630 receives and optionally displays readings of the pH, dissolved oxygen concentration, and dissolved carbon dioxide concentration of the first medium in the extracapillary space. Optionally, the computer 630 can control any of the controllable elements, such as pumps or valves, in the cell culture system 600. If a multiple reactor 550 is used, a set of sensors similar to sensors L, M, N can be provided in the panel 408 of each second element 402.

[0061] One or more gases are provided to the reactor 450 from one or more compressed gas tanks 602 or other gas sources. In the example shown, there are compressed gas tanks 602 for oxygen, carbon dioxide, nitrogen, and air. The gases flow through piping to a gas mixer 604, or optionally, a mass flow controller, which creates a gas blend. The gas blend passes through piping, optionally past one or more in-line sensors 606, through an optional in-line gas filter 608, and to the cap 430 of the reactor 450. In the example shown, in-line sensors 606 I and J sense the oxygen concentration and temperature of the gas blend. From the cap 430, the gas blend flows through the gas transfer membrane 102b inside the second reactor 450 to the opposite cap 430. Optionally, the gas blend then flows through more piping, optionally another in-line gas filter 608, and one or more optional additional in-line sensors 606 to an optional off-gas analyzer 610. In the example shown, an in-line sensor K senses the temperature of the gas blend exiting the second reactor 450. In the example shown, an off-gas analyzer measures the carbon dioxide and oxygen concentrations of the gas blend.

[0062] Optionally, flow control valves can be provided before or after reactor 450 to control the flow rate of gas through second reactor 450. When a composite reactor 550 is used as second reactor 450, piping to and from composite reactor 550 can be connected to gas perfusion manifold 534 and gas perfusion header 536, optionally with control valves 538, as described in connection with FIG. 10B, so that gas flow can be controlled to one or more individual elements 402.

[0063] The second medium is provided to the reactor 450 from the spinner flask 616 or another medium reservoir, such as a bottle or bioprocess bag. Optionally, the second medium can be added to the spinner flask 616 from a bottle 620 connected to a peristaltic pump 618. Optionally, the second medium can also be removed from the spinner flask 616 by another peristaltic pump 618 connected to another bottle 620. The other peristaltic pump 618 causes the second medium to flow into a recirculation loop between the spinner flask 616 and the perfusion membrane 102a of the reactor 450. The second medium flows from the spinner flask 616 to the upstream cap 430, through the perfusion membrane 102a inside the reactor 450, and to the downstream cap 430. The second medium then flows from the downstream cap 430, through further tubing, and back to the spinner flask 616. Optionally, the second medium in the recirculation loop may also flow through one or more in-line sensors 606 upstream and / or downstream of the reactor 450. In the example shown, in-line sensors 606A, B, C, and D sense the dissolved oxygen concentration, pH, dissolved carbon dioxide concentration, and temperature of the second medium entering the reactor 450. In the example shown, in-line sensors 606E, F, G, and H sense the dissolved oxygen concentration, pH, dissolved carbon dioxide concentration, and temperature of the second medium exiting the reactor 450. A filtered gas vent 612 is provided to allow air to move into or out of the second medium system while preventing contamination of the second medium. Optionally, the flask or bottle can be replaced with a variable volume bioprocess bag. In some cases, the filtered gas vent 612 is not required because air does not need to move into or out of the bioprocess bag as the volume of fluid in the bioprocess bag changes.

[0064] Optionally, flow control valves can be provided before or after the reactor 450 to help control the flow rate of the second medium through the reactor 450 or the pressure of the second medium at the perfusion membrane 102a. When a composite reactor 550 is used as the reactor 450, piping to and from the composite reactor 550 can be connected to a liquid perfusion manifold 530 and a gas perfusion header 532, optionally with control valves 538, as described in connection with FIG. 10A , allowing the flow or pressure of the second medium to be controlled for one or more individual second elements 402. Optionally, or in addition, the pressure of the second medium at the perfusion membrane 102a (and the corresponding pressure of the first medium in the ECS) can be varied by raising or lowering a spinner flask 616 or other second medium reservoir relative to the reactor 450, 550.

[0065] Optionally, the in-line sensors 606 for pH, carbon dioxide concentration, and oxygen concentration may be optical in-line sensors. For example, an optical in-line sensor manufactured by PreSens has a fiber optic cable 628 connected to a fiber optic meter 634, as shown in FIG. 13. The fiber optic meter 634 is connected to a computer 630 via a wire 635. Other in-line sensors 606, such as a temperature-sensing thermocouple, may also be connected to the computer 630. As shown in FIG. 13, the wire 635 from the thermocouple may be coupled to the fiber optic meter 634 before being connected to the USB port.

[0066] The illustrated cell culture system 600 is a small-scale system. Larger systems can be fabricated, for example, to operate a larger, complex reactor 550 or to operate multiple reactors 450 or multiple complex reactors 550 in parallel. Larger or other systems can use different sizes or types of equipment or different arrangements of conduits, valves, flow control devices, sensors, pumps, heaters, or other devices than the cell culture system 600, while achieving similar functionality. The computer 630 can be a general-purpose computer or any type of programmable controller, such as a programmable logic controller (PLC). The computer 630 can be a single, standalone computer located near the reactors 450, 550. Optionally, the computer 630 can be composed of multiple components, some of which are remotely located and connected to local components via data cables, wireless communication, or the Internet. In addition to operating the reactors 450, 550, the computer 630 can record data, communicate results, send error messages or alerts, or perform other functions.

[0067] The second medium circulates outside the reactor 450; in contrast, circulation of the first medium is preferably minimized. Optionally, there is no circulation of the first medium outside the reactor 450. The first medium is different from the second medium as determined by the pore size of the perfusion membrane 102a. In some examples, the perfusion membrane 102a can have a molecular weight cut-off (MWCO) selected from the range of 5,000 to 250,000 Da or more. Optionally, the perfusion membrane 102a can have a pore size in the range of 0.04 to 0.5 microns, or in the range of about 0.1 to 0.2 microns. Membranes with pore sizes in the range of 0.04 to 0.5 microns are useful, for example, when using a second medium containing large molecules, such as albumin or other proteins, intended for delivery to cells. Cells, viruses, and molecules above the MWCO are retained in the first medium. Large molecules retained in the extracapillary space include, for example, growth factors and proteins. In some instances, the first medium contains large molecules, such as growth factors, that substantially affect the cost of the cell culture process. Retaining these large molecules in the first medium and minimizing or eliminating circulation of the first medium outside of reactor 450 reduces the amount of these large molecules required to operate the process. Retaining large molecules or other products of the process in a limited volume of the first medium can also aid in the collection of these products. Because membranes 102 exclude cells, they also prevent contamination by bacteria, for example, in the gas or second medium portion of the system, from contaminating product cells or product-producing cells in the extracapillary space.

[0068] Despite the separation of the first medium into the extracapillary space by membrane 102, the second medium and gas can affect the first medium. For example, flow of gas or second medium through membrane 102 can be used to cool (or heat) the first medium. The carbon dioxide concentration and pH of the first medium can be affected, for example, by an acid, base, or buffer provided to the first medium through perfusion membrane 102a, or by adding or removing carbon dioxide from the first medium through gas transfer membrane 102b. Some nutrients small enough to pass through the pores of perfusion membrane 102a can also be provided to the first medium from the second medium. Small cell respiration products can be inhibitory and can also be removed from the first medium by diffusion through perfusion membrane 102a into the second medium.

[0069] FIG. 12 shows an alternative gas system 620 for the cell culture system 600 of FIG. 11. In the alternative gas system 620, the gas mixture travels from the gas mixer 604 to a pressure break 622. The pressure break 622 may be a T-junction with a filtered gas vent 612 in one arm. Excess gas is vented from the alternative gas system 620 to the atmosphere through the pressure break 622. Optionally, the vented vapor can be controlled with a valve. Optionally, a pressure regulator or other flow control valve can be used instead of or in addition to the pressure break 622. A gas pump 624 downstream of the reactor 450 draws some of the gas mixture under vacuum through the gas transfer membrane 102b. A rotameter 626, or optionally a mass flow meter, measures the flow of the gas mixture before it travels to the off-gas analyzer 610. Optionally, the rotameter 626 can be used to provide a signal for use in a feedback or other control loop connected to the gas pump 624, the pressure break 622, or both. The pressure break 622 and gas pump 624 can be selectively configured so that the inside of the gas transport membrane 102b is under partial vacuum or pressure at the upstream end and under partial vacuum or pressure at the downstream end. Gas, such as oxygen, can flow from the gas transport membrane 102b to the first medium if the partial pressure of the gas inside the gas transport membrane 102b is sufficient relative to the concentration of the gas in the first medium. Gas can be delivered to the first medium even if the total pressure of the gas mixture inside the gas transport membrane 102b is less than atmospheric pressure or less than the pressure of the first medium surrounding the gas transport membrane 102b.

[0070] In some cases with the gas system shown in FIG. 11, the total pressure of the gas mixture or the partial pressure of one or more specific gases inside the gas transport membrane 102b can cause undesirable results. For example, the pressure of the gas mixture inside the gas transport membrane 102b can be sufficient to cause one or more gases to form bubbles in the first medium. While the total pressure of the gas mixture can be adjusted, undesirable pressures may be required to provide the desired flow rate of the gas mixture. Changing to the alternative gas system 620 in FIG. 12 allows for different combinations of gas pressure and flow rate to better suit a particular process. In the case of the composite reactor 550, the control valve 538 shown in FIG. 10B can also be used to control the flow of gas through the individual second elements 402.

[0071] Erythroid progenitor cells tend to expand and differentiate into red blood cells more rapidly under low oxygen tension, and some other cells also prefer low oxygen. High oxygen transport capacity (kLa) is required to support large and / or rapidly proliferating cell populations. However, oxygen transport is preferably not driven by having localized areas of high dissolved oxygen concentration (generally relative to the dissolved oxygen concentration within the reactor) in direct communication with the cells in the ECS. The gas transport membrane 102b can help maintain a low-oxygen environment in the ECS, which still supports high cell densities, by providing oxygen transport that is well-controlled and generally evenly distributed throughout the extracapillary space. Alternatively, the gas transport membrane 102b can be used to provide a high dissolved oxygen concentration (i.e., 10% solubility or greater) throughout a majority of the ECS volume (i.e., 50% or greater, 75% or greater, or 90% or greater).

[0072] Cells and other products can be harvested from the reactor 450, 550 through the fitting 164 on the top plate 436 or through the panel 408. If the fitting 164 on the top plate 436 is used for harvesting, the fitting 164 is positioned adjacent to the periphery of the top plate 436. In either case, the reactor 450, 550 is rotated so that the fitting 164 is positioned on the bottom of the ECS. The opposing fitting 164 on the top of the bioreactor 450, 550 is connected to a valve and air filter. When the valve is opened, the ECS containing the cells or other products is discharged from the reactor 450, 550. The discharge fitting 164 can be attached to tubing, which transports the contents of the ECS to a receiving vessel, such as a bioprocessing bag or another reactor 450, 550. If the receiving vessel is not below the reactor 450, 550, a pump or compressed air can be used to transport the contents of the ECS to the receiving vessel. Optionally, harvesting can involve transferring cells from one reactor 450, 550 to another reactor 450, 550 and can be triggered by a measurement, such as VCD or an indicator of cell differentiation or cell metabolism.

[0073] The ECS may be initially filled by adding the inoculum and first medium through the bottom fitting 164 while opening the valves and venting air through the top fitting 164. After a measured amount of inoculum and first medium substantially equal to the volume of the ECS has been added, or when liquid is detected in the top fitting 164, the valves are closed and filling stops.

[0074] Optionally, a collection layer can be added to the reactor 450, 550 and configured to provide one or more separation steps or to allow collection of products other than the cells themselves. For example, in the case of cultured red blood cells, a suspension of cells can be removed from the reactor and separated into a) nucleated cells and b) a mixture of enucleated cells and nuclei in a first step. The mixture can be separated in a second step, with the enucleated cells being extracted from the nuclei (which become waste). Optionally, the collection layer is configured to perform a portion of the first step, i.e., the nucleated cells are selectively retained within the reactor, while the enucleated cells and nuclei are selectively extracted from the reactor. The degree of separation need not be complete to be useful; a second separation of enucleated cells from nucleated cells can occur outside the reactor. In another example, if the bioreactor 450, 550 is used to produce virus intracellularly, the produced virus can be extracted via the collection layer, while the cells are retained in the bioreactor. In another example, if the cells in the bioreactor 450, 550 are used to produce a protein or other product, the product can be extracted through a collection layer while the cells are retained in the bioreactor 450, 550.

[0075] The perfusion membrane may be part of a recirculation loop that includes a bioprocess bag or other reservoir of second medium (e.g., spinner flask 616 shown in FIG. 11). The reservoir preferably has a variable volume. In this way, additional liquid, such as a pH-adjusting solution or a solution containing cytokines or other reactants, can be added as needed without the need to simultaneously remove liquid from the recirculation loop. In batch operation, the extracapillary space (ECS) and reservoir are filled with second medium before the start of the run, and no additional second medium is added during the run. In fed-batch operation, the ECS and reservoir are filled to an initial volume before the start of the run, but at one or more later time points, additional second medium is added to the reservoir, for example, from a second bioprocess bag or other container (e.g., bottle 620 shown in FIG. 11), and no second medium is removed. In continuous or feed-and-bleed operation, the ECS and reservoirs are filled to an initial volume before the start of the run, and the second medium is added to and removed from the reservoirs during the run (which can include, for example, continuously, in pulses, or at other discrete time intervals; at a constant rate, or at a rate that varies with time), as described, for example, in connection with FIG. 11. The second medium can be added to or removed from the reservoirs directly or by adding or removing medium from a recirculation loop. The reservoirs are preferably heated to the same temperature as the reactors 450, 550 (i.e., 25-50°C, or 37°C). For example, the reservoirs can be placed in an incubator with the reactors 450, 550 or in a separate heated area. The second bioprocess bag or other container containing the second medium for a fed-batch or feed-and-bleed process can be maintained at room temperature during the run or can be refrigerated (i.e., 4-8°C).

[0076] In another mode of operation, multiple reactors 450, 550 can operate using separate ECSs and their membranes 102 linked together. For example, the secondary medium perfusion and / or gas perfusion ports of multiple reactors 450, 550 can be linked in parallel. Due to the membranes 102, cells in the ECS of one reactor 450, 550 cannot enter the ECS of another reactor 450, 550. In this way, cells of the same type but derived from different donors or intended for different patients can be cultured simultaneously but separately. While each cell population differs in some respects, the cell population dynamics are similar enough that some secondary medium perfusion or gas delivery system components can be shared. Optionally, the flow of secondary medium or gas to each individual reactor 450, 550 can be regulated in a manner similar to the control of the individual secondary elements 402 in the composite reactor 550 described above.

[0077] The membrane 102 provides perfusion, i.e., delivery of substances in a dispersed manner. The membrane 102 also provides retention, i.e., cells are retained within the ECS. The membrane 102 can also retain selected compounds within the ECS. This can reduce the amount of costly components, such as growth factors, required because the ECS is smaller than the entire bioreactor 450, 550 and its recirculation loop. As these selected compounds are consumed or decompose, more of the selected compounds can be added directly to the ECS without adding the entire medium. In this way, compounds that decompose at different rates can be added at appropriate rates.

[0078] The reactor 450, 550 can be cooled (or heated) by cooling the second medium or gas. This can make the reactor 450, 550 more accessible than, for example, wrapping the reactor in a cooling jacket. Furthermore, the use of a cooling jacket increases the reactor diameter, so there can be a temperature difference between the core and the jacket. By delivering cooling through the membrane 102, heat is removed from the center of the reactor 450, 550.

[0079] Mixing in the ECS disrupts the boundary layer around the membrane 102. This disruption can increase the transport rate associated with the membrane 102, but also tends to equalize the transport rates in different portions of the reactor 450, 550. The first medium moves, which helps to equalize the delivery of the gas and second medium components. Movement of the first medium can also be provided by moving the entire reactor 450, 550, for example, by rotating about any axis, rocking back and forth, inverting, rotating at an angle, or another movement that changes the direction of the gravity vector relative to the reactor 450, 550.

[0080] By moving reactors 450, 550, the surface area of ​​the gas transport membrane can be reduced relative to a reactor without mixing in the ECS. The reduced surface area of ​​the gas transport membrane 102b, in turn, facilitates culturing cells in suspension and harvesting of cells and cell products. In some examples, the volume of the ECS is reduced by 1 mm of surface area of ​​the gas transport membrane 102b. 2 1-20, 2-20, or 2-10 mm per 3 (ECS volume to gas transfer surface area ratios of 1-20 mm, 2-20 mm, or 2-10 mm). The length and surface area of ​​membrane 102 are measured within the ECS (i.e., only the length and surface area directly exposed to the ECS are considered) unless otherwise specified.

[0081] Harvesting the cells from the reactor 450, 550 can be accomplished by opening one or more fittings 164 on the top plate 436 and / or one or more panels 408, optionally attaching tubing to the one or more fittings 164, and draining the first medium from the ECS. Draining the first medium can be accomplished, for example, by orienting the reactor 450, 550 so that the drain fittings 164 are near the bottom of the reactor 450, 550, allowing the first medium to drain by gravity. In some examples, draining the ECS can recover 80% or more or 90% or more of the cells in the reactor 450, 550 before harvesting. Optionally, after draining the ECS, the ECS can be rinsed by adding a liquid (i.e., buffer or medium) to the ECS, optionally moving the reactor 450, 550 to mix the liquid, and draining the added liquid. The added liquid can range from 20-200% or more of the ECS volume. Rinsing the reactor 450, 550 can allow for additional cell recovery, particularly for semi-adherent cells, such as HEK cells. Rinsing the reactor 450, 550 can also be useful to aid in the transport of harvested cells from one reactor 450, 550 directly to a larger reactor 450, 550. In some instances, tubing is used to connect the ECSs of two reactors 450, 550. Cells harvested from one reactor 450, 550 are used to inoculate a larger reactor 450, 550 for further cell growth. In this case, the rinse agent can be the medium of the first culture medium used in the larger reactor 450, 550.

[0082] In some examples, movement of the first medium is provided by rotating the reactor 450, 550, optionally about an axis perpendicular to the membrane 102 and / or about a horizontal axis. The axis of rotation may pass through the reactor 450, 550, optionally through the center of the reactor 450, 550. For example, the reactor 450, 550 may be rotated as shown in Figures 8, 9A, and 9B. The rotation is preferably in both directions (i.e., clockwise and counterclockwise), e.g., rather than continuously or continuously (i.e., more than one or more than five rotations in either direction). Optionally, the reactor 450, 550 does not rotate more than one revolution before the direction of rotation is reversed. For example, the reactor 450, 550 may be rotated from a starting position in one direction by 45-720 degrees, or 90-360 degrees, or 120-240 degrees, and then rotated in the opposite direction by 45-720 degrees, or 90-360 degrees, or 120-240 degrees, optionally returning to the starting point. In some examples, the reactor 450, 550 may be inverted (i.e., rotated 180 degrees or more) or nearly inverted (i.e., rotated 120-179 degrees) before reversing the direction of rotation. The movement in either direction may be one continuous movement or a series of smaller movements. For example, the reactor 450, 550 may be rotated 180 degrees in six movements of 30 degrees each in one direction (or four movements of 45 degrees each, etc.), and then re-inverted in six movements of 30 degrees each in the opposite direction (or four movements of 45 degrees each, etc.). The movements described herein are typically performed in a repeating pattern, optionally returning to the starting position at the end of each pattern. Optionally, the rotation axis may be vertical. However, a horizontal rotation axis, or at least an oblique rotation axis, can enhance, for example, the movement or suspension of cells or other solids in the first medium by inversion, partial inversion, or other gravity-induced movement.

[0083] Without intending to be limited by theory, accelerating and decelerating the reactor 450, 550 and / or reversing the rotation can help increase the movement of the first medium relative to the membrane 102. After a period of sustained rotation in one direction at a constant speed, the first medium may tend to move in the same direction and at the same speed as the membrane 102. Frequently accelerating and decelerating the reactor 450, 550 and / or reversing the direction of the reactor 450, 550 can help provide and / or increase the difference in the velocity of the first medium relative to the membrane 102. This effect is distinct from rocking the bioreactor to create waves along the free surface of the bioreactor because the reactor 450, 550 typically operates substantially without a continuous gas phase or headspace (i.e., less than 5% of the ECS volume is filled with a continuous gas phase); the reactor 450, 550 does not have a length, measured in a horizontal direction perpendicular to the axis of rotation, that is significantly greater than the depth of the first medium; and / or any free surface of the liquid first medium may not extend along the entire length of the reactor 450, 550. In some examples, the reactor 450, 550 may be circular or a regular polygon (i.e., a square, pentagon, hexagon, or octagon with sides of equal length) in cross section perpendicular to the axis of rotation, or, if not a regular polygon (i.e., rectangular), may have a length-to-depth ratio of 3 or less, or 2 or less, in cross section perpendicular to the axis of rotation. Rotation of the reactor 450, 550 also differs from, for example, a flask on a shaker table, in which the medium is caused to rotate against the flask wall, but there is no rotation of the flask against the flask's central vertical axis. Such movement of a shaker flask typically requires undesirable speeds and forces to be generated in combination with the membrane 102, and relies on the creation of a liquid film in contact with a large continuous gas phase. Roller bottle movement may be slower, but it also relies on the creation of a large area film with a free surface, which does not occur to the same extent in the reactor 450, 550 due to the lack of headspace in the reactor 450, 550.

[0084] The rotational speed and / or acceleration or deceleration of the reactor 450, 550 can be selected to provide a desired shear rate or tangential velocity at a selected location, e.g., around the periphery of the ECS. Tangential velocity varies with rotational speed and diameter. For example, an 85 mm diameter reactor 450 with 0.2 L of ECS was moved in a repeating pattern of rotating in one direction, then rotating in the opposite direction back to the starting point. The maximum rotational speed in the pattern was 5 rpm. The ramp-up and ramp-down times (transition times between maximum rotational speed and stopping of rotation) were minimal, i.e., approximately 0.4 seconds, with no dwell time between direction changes. To provide the same maximum tangential velocity, a 200 mm diameter reactor 450 with a 1.2 L ECS volume was moved following the same pattern but using a maximum rotational speed of 2.125 rpm. The rotational speeds described herein are calculated by comparing the product of their rotational speed and diameter, i.e., S1 * D1=S2 * D2 (where S is the rotation speed and D is the reactor diameter) can be similarly adjusted for use with reactors of different diameters.

[0085] Alternatively, or in addition, reactor movements can be compared by considering reversal rates. For example, a reactor rotated 180 degrees back and forth at 5 rpm will change direction approximately once every 6 seconds, regardless of its diameter, assuming minimal ramp-up, ramp-down, and dwell times (i.e., ramp-up and ramp-down times of 0.1-0.5 seconds, dwell times of 0.0-0.5 seconds). Another reactor rotated 360 degrees back and forth at 10 rpm has the same reversal rate, approximately once every 6 seconds. A reactor rotated 360 degrees at 5 rpm has a lower reversal rate, approximately once every 12 seconds.

[0086] As the reactor diameter increases, the degree of rotation between inversions can be reduced while maintaining tangential velocities similar to those of smaller reactors, providing a) an inversion frequency and / or b) a rotation distance around the reactor similar to those of smaller reactors. However, it may be desirable to occasionally invert or nearly invert the reactor 450, 550 to prevent cells from collecting on the bottom of the reactor 450, 550. Optionally, the movement pattern can include a first rotational movement back and forth of less than 180 or 120 degrees to provide a first medium movement against the membrane 102, periodically interrupted by a second rotational movement of at least 120 or 180 degrees back and forth to prevent cell settling. Alternatively, small movements in each direction (i.e., movements less than 180 or 120 degrees) are unequal but combined in a pattern such that the reactor is periodically inverted or nearly inverted. For example, the reactor 450, 550 can be rotated 90 degrees clockwise and 60 degrees counterclockwise six times, then 60 degrees clockwise and 90 degrees counterclockwise in a repeating pattern. Alternatively, movement in one direction can be broken down into a series of smaller movements in the same direction, as described further above. Stopping and restarting rotation in one direction may provide less mixing than reversing direction, but stopping and restarting rotation in one direction can provide sufficient movement of the first medium against the membrane. Optionally, when using the stop-and-restart motion option in one direction, the tangential velocity can be doubled for instances with multiple direction changes. Optionally, the movement of the reactor 450, 550 includes 5-30, 8-30, or 10-30 accelerations and decelerations, direction changes, or stops and restarts in the same direction per minute.

[0087] In at least some instances, oxygen transfer from the gas transport membrane 102b to the ECS becomes a limiting factor in increasing cell growth rate or density. In various tests, oxygen transfer from the gas transport membrane increased with the back and forth rotation of the reactor 450, 550 as described above relative to the operation of the impeller inside the ECS of the reactor 450, 550.

[0088] Successful operation of an 85 mm diameter reactor similar to reactor 450 oriented in Figure 8 was achieved with rotational speeds of 2-20 rpm and translations back and forth over a range of motion from 180-720 degrees. In some instances with 180 and 360 degrees of translation back and forth, the kLa of oxygen transfer to deoxygenated water increased with speeds up to about 7 or 8 rpm and then became somewhat constant at higher speeds. The kLa of oxygen transfer to cells growing in culture is believed to be higher than transfer to deoxygenated water. In cell growth experiments, rotation back and forth 180 degrees at speeds of 5-13 rpm demonstrated sufficient oxygen transfer to support high cell densities, e.g., an ECS of approximately 58 million CHO cells / mL, at 11-13 rpm, with 70% oxygen being entrained in the air flowing through the 8-layer gas transport membrane 102b. ECSs of over 70 million or even over 100 million CHO cells / mL were reached at 15 rpm, with a limit of 80% oxygen being entrained in the air flowing through the 15-layer gas transport membrane 102b. In other tests, increasing the speed to 18 and 20 rpm provided further increases in oxygen transfer rate. However, the maximum speed is not always used, as maximum oxygen transfer rate is not required for all cell growth processes; cells may reach maximum density for reasons other than oxygen transport, or shear forces may be limiting for some cells. For example, for cells growing to lower cell densities, i.e., CD34+ cells, rotation back and forth at 360 degrees and a speed of 2.5 rpm provided sufficient oxygen transfer. In another study, T cells were grown to a cell density of 15 million cells / mL using a speed of 2.5-5 rpm, and no additional oxygen was required in the gas stream. Successful operation of a 200 mm diameter reactor similar to Reactor 450, oriented as in Figure 8, was achieved with rotation speeds of 2-7 rpm and a range of motion of 180-360 degrees back and forth.

[0089] In an experiment using a 200 mm diameter reactor, oxygen distribution through the reactor was measured using sensors on the top plate 436 of the reactor 450 near the inlet of the gas transport membrane, near the center of the reactor, and near the outlet of the perfusion membrane. A 360° back-and-forth movement pattern at 2.5 rpm was sufficient to maintain an oxygen threshold of approximately 12.5% ​​solubility (i.e., 59.5% of the oxygen concentration in water at equilibrium with air) as measured by the first and third sensors, but the oxygen level determined by the center sensor decreased as cell density increased. The difference in oxygen concentration between the three sensors, particularly between the sensor in the center of the reactor and the other two reactors, decreased when going from a) 2.5 rpm, 360° back-and-forth, to b) 3 rpm, 360° back-and-forth, to c) 5 rpm, 360° back-and-forth, and to d) 5 rpm, 180° back-and-forth. Transfer patterns c) and d) were considered to be substantially better at transporting oxygen to the center of the reactor.

[0090] Based on various examples, it is estimated that for reactors of similar size (i.e., diameters ranging from 50-300 mm or 75-250 mm), rotation at speeds at least 50% less and at least 100% greater than the values ​​described herein may also result in successful operation. The amount of travel between inversions is optionally 360 degrees or less, 270 degrees or less, or 180 degrees or less for reactors of similar size (i.e., diameters ranging from 50-300 mm or 75-250 mm). For reactors of substantially different sizes, appropriate parameters can be developed using the above scaling guidelines.

[0091] In these examples, there was essentially no dwell time between reversals of direction of rotation. Optionally, there may be a short dwell time, e.g., in the range of 0.1 to 1 second. Extending the dwell time, and / or velocity ramp-up or ramp-down time, can help reduce stress on the membrane 102 and cells. The reactor was generally described for reactor 450, e.g., as shown in FIG. 8 and further described below in the Examples below. The arrangement for moving the reactor was generally as shown in FIGS. 8, 9A, or 9B.

[0092] Cells are typically grown outside the membrane 102 in the ECS of the reactor 450, 550. The cells can be maintained in the ECS of the reactor 450, 550, i.e., optionally without circulation through any components of the system external to the reactor 450, 550. The movement of the reactor 450, 550 helps facilitate material transfer between the membrane 102 and the ECS, allowing for low packing densities, e.g., 20% or less, 15% or less, or 10% or less, calculated based on the volume of the membrane 102 divided by the internal volume of the reactor 450, 550 (i.e., the volume of the ECS added to the volume of the membrane 102). In various examples described below, the reactor 450, 550 has an overall packing density (calculated as the volume of the cumulative gas transport membrane and liquid transport membrane passing through the ECS divided by the volume of the ECS) of about 8-12%. Low packing densities increase the volume available for cell growth (i.e., ECS volume) for a given reactor size and / or aid in collection. In various examples of suspension cell culture, the majority (i.e., 90% or more) of cells were collected from reactors with packing densities of approximately 8% at viable cell densities (VCDs) of up to 58 million cells / mL. In contrast, in bioreactors with high packing densities (i.e., 30-40% or more), cells tend to become trapped between the membranes even when not attached and cannot be effectively collected.

[0093] In various tests, the inventors observed that gas transport through membrane 102 was more limited than the transport of dissolved materials, such as glucose or lactate, to or from the first medium.

[0094] Optionally, the movement pattern causes the membrane 102, e.g., gas transport membrane 102b, to move across the top surface of the reactor 450, 550. Combined with the liquid pressure in the ECS exceeding the gas pressure inside the gas transport membrane 102b, this minimizes or eliminates any headspace. For example, an initial headspace may be present upon filling the reactor 450, 550. Bulk phase gas may be removed from this initial headspace through the membrane 102, thereby reducing or eliminating the headspace. Similarly, gases added to or generated in the reactor during operation are prevented from forming a significant headspace. Optionally, less than 5%, or less than 2%, or less than 1% of the volume of the ECS is filled with a continuous gas phase. The presence of headspace tends to lead to bubbles and / or foam, which are detrimental to cell growth.

[0095] As shown in the examples below, mixing by moving the reactor combined with low membrane potting or packing density allows cells to reach high viable cell densities (VCDs) in suspension. In these examples, over 90% of the cells can be collected by simply draining the reactor. Furthermore, mixing by moving the reactor in the examples described below results in generally uniform conditions throughout the reactor. A variety of different cell types can be grown.

[0096] Without intending to be bound by theory, reactor movement with frequent changes in speed and / or direction can result in sustained transient flow conditions in the reactor. The power required to move the bioreactor reaches a peak or maximum during or near periods of bioreactor acceleration, deceleration, or direction reversal. During these periods, or near the beginning of one of these periods, the required power can reach a minimum. Average power can be defined as the absolute value of the minimum power plus the absolute value of the peak power (i.e., ignoring direction or polarity) divided by 2. Because fluid in the ECS can become entrained in the membrane movement, the applied power may exceed the average power for periods longer than the acceleration, deceleration, or reversal portion of the movement cycle. In some instances, the applied power can exceed the average power for a portion of the material, i.e., for 20% or more, 25% or more, or 33% or more, optionally up to 50% of the movement cycle. Relatively long periods of high power can increase transient flow or mixing in the ECS. Another theory is that the transport cycle may not have a sufficient period of unidirectional motion for the liquid velocity to reach the same velocity in the same direction as the membrane in at least a portion of the reactor, e.g., near the periphery of the reactor. Thus, for most (i.e., 50% or more, 70% or more, or 90% or more) of the transport cycle in at least a portion of the reactor, e.g., near the periphery of the reactor, there may be a velocity difference between the membrane and the surrounding liquid. Alternatively, or in addition, the velocity difference may be higher near the periphery of the reactor than near the center of the reactor, which may cause shear (and thus mixing) between the periphery of the reactor and the center of the reactor.

[0097] In the following examples, k L a is for oxygen unless otherwise specified.

[0098] Example 1 Reactors similar to reactor 450 described herein and shown in FIG. 8 were fabricated with an ECS volume of approximately 200 mL, with slight variations in ECS volume between individual reactors. The reactors had a roughly cylindrical ECS with an internal diameter of approximately 85 mm. The reactor had 15 layers of gas transport membranes, with each layer having 40 membranes spaced at a center-to-center distance (pitch) of 0.8 mm (600 membranes total). The gas transport membranes were OXYPLUS™ PMP skin membranes from 3M and had an external diameter of approximately 0.38 mm. In use, the reactor was oriented with the membranes in a vertical plane and rotated back and forth on a horizontal axis, as shown in FIG. 8. The total surface area of ​​the gas transport membranes was 58,600 mm. 2 The ECS volume to gas transport membrane surface area ratio was 3.4 mm. The reactor also had eight layers of perfusion (liquid transport) membranes, each layer having 18 membranes spaced at a center-to-center distance (pitch) of 2 mm (144 membranes total). In some examples, the perfusion membranes were PES membranes with a molecular weight cut-off (MWCO) of approximately 10 kDa and an outer diameter of approximately 1.2 mm. In some examples, the perfusion membranes had a pore size of approximately 0.1 microns. In the examples described further below, the reactor according to Example 1 has 10 kDa perfusion membranes unless otherwise noted. The membrane layers were alternating with two layers of gas transport membranes separating each layer of perfusion membrane.

[0099] Example 2 A reactor similar to that of Example 1 was constructed with eight layers of gas transport membranes, each layer having 40 membranes spaced at a center-to-center distance (pitch) of 0.8 mm (320 membranes total). The gas transport membranes were OXYPLUS™ PMP skin membranes from 3M and had an outer diameter of approximately 0.38 mm. In use, the reactor was oriented horizontally (membranes in the vertical plane) and rotated back and forth on a horizontal axis, as shown in Figure 8. The total surface area of ​​the gas transport membranes was 31,200 mm. 2The ECS volume to gas transport membrane surface area ratio was 6.4 mm. The reactor also had eight layers of perfusion (liquid transport) membranes, each layer having 18 membranes spaced at a 2 mm center-to-center distance (pitch) (144 membranes total). The perfusion membranes were PES membranes with an approximately 10 kDa molecular weight cutoff (MWCO) and an outer diameter of approximately 1.2 mm. In some examples, the perfusion membranes had a pore size of approximately 0.1 microns and had different outer diameters and membrane numbers, but the same surface area as the 10 kDa membranes. In the examples described further below, the reactor according to Example 2 had 10 kDa perfusion membranes unless otherwise noted. The membrane layers were configured with alternating layers of gas transport membranes separating each layer of perfusion membrane.

[0100] Example 3 A reactor similar to Reactor 450 described herein and shown in FIG. 8 was fabricated with an ECS volume of approximately 1,200 mL. The reactor had an ECS internal diameter of approximately 200 mm. The reactor had eight layers of gas transport membranes, each layer having 106 membranes spaced at a center-to-center distance (pitch) of 1.08 mm (848 membranes total). The reactor also had eight layers of liquid transport membranes, each layer having 48 membranes spaced at a center-to-center distance (pitch) of 2.35 mm (384 membranes total). The liquid transport membranes were PES membranes with a molecular weight cutoff (MWCO) of approximately 10 kDa and an outer diameter of approximately 1.2 mm. The gas transport membranes were OXYPLUS™ PMP skin membranes from 3M with an outer diameter of approximately 0.38 mm. In use, the reactor was oriented with the membranes in a vertical plane and rotated back and forth on a horizontal axis, as shown in FIG. 8. The membrane layers consisted of alternating layers of gas transport membrane separating each layer of perfusion membrane.

[0101] Example 4 A reactor similar to the composite reactor 550 described herein and shown in Figure 9A or 9B was made with an ECS volume of approximately 1 L. The reactor had a stack of 5 of the reactors of Example 2. In use, the reactor was oriented with the membrane in the vertical plane and rotated back and forth on a horizontal axis 165, as shown in Figure 9A or 9B.

[0102] Example 5 A reactor similar to the composite reactor 550 described herein and shown in Figure 9A or 9B was made with an ECS volume of approximately 5.5 L. The reactor had a stack of 5 of the reactors of Example 3. In use, the reactor was oriented with the membrane in the vertical plane and rotated back and forth on a horizontal axis 165, as shown in Figure 9A or 9B.

[0103] Example 6 A reactor similar to the composite reactor 550 described herein and shown in Figure 9A or 9B was made with an ECS volume of approximately 10 L. The reactor had a stack of 9 of the reactors of Example 3. In use, the reactor was oriented with the membrane in the vertical plane and rotated back and forth on a horizontal axis 165, as shown in Figure 9A or 9B.

[0104] Example 7 A reactor similar to that of Example 1 was constructed with 14 layers of gas transport membranes, each layer having 40 membranes spaced at a center-to-center distance (pitch) of 0.8 mm (560 membranes total). The gas transport membranes were OXYPLUS™ PMP skin membranes from 3M and had an outer diameter of approximately 0.38 mm. In use, the reactor was oriented horizontally (membranes in the vertical plane) and rotated back and forth on a horizontal axis 165, as shown in Figure 8. The total surface area of ​​the gas transport membranes was 54,600 mm. 2 The ECS volume to gas transport membrane surface area ratio was 11.2 mm. The reactor also contained six layers of perfusion (liquid transport) membranes, each layer containing seven membranes spaced at a center-to-center distance (pitch) of 6.5 mm (42 membranes total). The perfusion membranes were PES membranes with a pore size of approximately 0.1 μm and an outer diameter of approximately 2.6 mm. The membrane layers were alternating with two layers of gas transport membranes separating each layer of perfusion membrane.

[0105] Example 8 k in the initial deoxygenated water La (volumetric oxygen transfer coefficient) was measured for the reactors according to Examples 2 and 3. The results are shown in Table 1. "Linear velocity" is calculated based on the rpm and reactor diameter. "Rotation angle" is the angle of rotation back and forth. "Reversals per minute" is the number of rotational direction changes per minute, estimated by the rotation speed and rotation angle. The column "Difference" shows the k measured by a sensor near the inlet of the gas transport membrane. L a and k measured by a sensor near the outlet of the gas transport membrane L The difference between the two measurements was calculated by dividing the average of the two measurements. In the 0.2-L reactor (Example 2), increasing the rotation speed increased k L As a increased, the difference between 5 and 7 rpm decreased, but then k L As a increases, the reduction in the difference becomes smaller. [Table 1]

[0106] Example 9 kLa was measured for reactors according to Examples 1, 2, and 3 when used to grow CHO cells. The results are shown in Table 2. "Rotation" is the rotation speed. "Linear velocity" is calculated based on the rpm and reactor diameter. "Rotation angle" is the angle of forward and backward rotation. "Turns per minute" is the number of rotational direction changes per minute, estimated by the rotation speed and rotation angle. In both examples shown in Table 2, the bioreactor was able to maintain an oxygen set point of 12.5% ​​solubility (i.e., 50% of the oxygen concentration of water at equilibrium with the atmosphere) in the ECS throughout the batch process, until the end of the period of glucose metabolism, and up to a viable cell density (VCD) of over 20 million cells / mL ECS. k L a was measured near the end of each run. L The VCD at which a was measured is expressed in million cells / mL in parentheses in the last column of Table 2. [Table 2]

[0107] Example 10 Oxygen readings were compared between three sensors placed in different parts of the head plate of a 1.2-L reactor according to Example 3. Sensor 1 was placed near the inlet of the gas transport membrane. Sensor 2 was placed near the inlet of the liquid transport membrane. Sensor 3 was placed near the center of the bioreactor, along the gas transport membrane. The oxygen flow was adjusted to provide 12.5% ​​solubility at Sensor 1. The results are shown in Table 3. At 2.5 rpm / 360°, the difference in oxygen concentration at different locations was high at 8.85%, with an average noise of ±0.48% on each sensor. Increasing the rotation to 5 rpm / 360° reduced the difference between sensors to 7.6%, and the noise to ±0.13%. Finally, changing the rotation angle to 5 rpm / 180° reduced the difference to 3.8%, and the noise to ±0.06% of the reading. Although all three movement patterns produce useful oxygen transfer overall, the oxygen concentration at sensor 3 for 2.5 rpm / 360° may indicate that cell growth will not be strong throughout the ECS. Based on other examples described herein, it is predicted that the results for 2.5 rpm / 360° can be substantially improved if the rotation angle is reduced to 180 degrees and provides 5 reversals per minute. [Table 3]

[0108] Example 11 Reactors were prepared as described in Examples 2, 3, 4, and 5 (identified in Table 1 by their volumes, which correspond to those given in the descriptions of Examples 2, 3, 4, and 5 above), except that the reactor in Run 9 had a liquid perfusion membrane with a 0.1 micron pore size rather than a 10 kDa membrane. The reactors were used to grow CHO cells in a batch process. In these examples, the term batch process indicates that the total volume of liquid medium used in the process was present at the start of the process. In particular, the ECS was filled with liquid medium. The perfusion system (including a circulating medium loop extending from the medium bag, through the liquid perfusion membrane, and back to the medium bag) was also filled with the same liquid medium. The volume of liquid medium in the reservoir was approximately twice the volume of the ECS. For example, in tests using the reactor of Example 2, approximately 600 mL of medium was provided: 200 mL in the ECS and 400 mL in the circulating medium loop.

[0109] The reactor was seeded with approximately 2 million cells / mL based on ECS volume and operated for more than three days (approximately 78 hours). The glucose concentration of the medium decreased from approximately 30 mM at the beginning of the process to near zero over approximately 72 hours. Cell metabolism appears to have been glucose metabolism for approximately 60-65 hours, followed by glucose and lactate cometabolism, and then lactate metabolism for the remainder of the run. Shake flask cultures were run as controls (aerated Erlenmeyer flasks, orbital shaker, 0.03 L total medium volume). The speed and degree of rotation before direction reversal, as well as various other results, are shown in Table 4. In Table 4, the "kLa Low" designation indicates that at some point in the run, the oxygen sensor near the periphery of the top plate used to control dissolved oxygen indicated that dissolved oxygen had dropped below the set point of 12.5% ​​solubility. The "kLa high" indication indicates that the oxygen sensor used to control dissolved oxygen showed 12.5% ​​solubility throughout the run (although oxygen sensors near the center of the reactor may have had lower readings for portions of the run). Cells continued to grow exponentially despite the "kLa low" conditions in some runs. However, oxygen readings at the inner periphery of the reactor can qualitatively indicate the effect of different patterns of rotation on oxygen transport. "td" is the cell VCD doubling time. "Y VCD ' is the yield of cells from glucose during exponential growth on glucose (determined from 0 to 60 hours) and expressed as million cells / mmol glucose consumed). Gluc " is the glucose consumption rate (pmol / (cell-day)) during the first 60 hours of the run. Lac " is the lactate production rate during the first 60 hours of the run. The ratio is the volume of the perfusion system divided by the volume of the bioreactor. Cell viability was greater than 90%, typically greater than 95%. [Table 4]

[0110] Figure 14 shows the VCD (measured based on ECS volume) for the various runs. The control flask run reached a VCD of 13 million cells / mL at 72 hours.

[0111] Example 12 The reactor was constructed as described in Example 2. The reactor was used to grow CHO cells. The ECS was initially filled with approximately 200 mL of liquid medium. A circulating liquid medium loop (perfusion system) extended from the medium bag, through the reactor's liquid perfusion membrane, and back to the medium bag. The perfusion system was initially filled with approximately 200 mL of the same liquid medium. After 48 hours of operation, fresh medium was added to the medium bag at a rate of 200 mL / day (and old medium was removed at the same rate). Between approximately 72 and 96 hours of operation, the rate of fresh medium addition and removal was gradually increased. The rate of fresh medium addition and removal was maintained at 600 mL / day from approximately 96 hours of operation until the end of the run (approximately 140 hours). Throughout the run, 1.9 L of medium was added to and removed from the medium bag. Total medium consumption was 2.3 L, including the first 200 mL of medium in the ECS and the first 200 mL of medium in the medium bag.

[0112] The reactor was seeded with approximately 2 million cells / mL based on the ECS volume. The speed and rotation before direction reversal were 11-13 rpm and 180 degrees, respectively. Compressed air was supplied to the gas transfer membrane for the first 24 hours, with occasional addition of carbon dioxide. From 24 to 78 hours, pure oxygen was blended into the air at increasing amounts, from 0 to 70% of the total gas flow rate, as needed to maintain an oxygen setpoint range of 10-15% solubility in the ECS (i.e., 50% of the oxygen concentration in water at equilibrium with the atmosphere). After 78 hours, the pure oxygen flow was maintained at 70% of the total air flow rate.

[0113] After 78 hours of operation, the VCD reached approximately 25 million cells / mL of ECS volume. Between approximately 60 and 96 hours, oxygen sensors on the periphery of the top plate indicated that the oxygen concentration dropped from 12.5% ​​solubility to undetectable amounts. However, cells continued to proliferate between 70 and 120 hours, with an average doubling time of approximately 34.3 hours, reaching a maximum VCD of approximately 589 million cells / mL of ECS volume at 120 hours. Cell viability remained above 90% during this period. Between 120 and 140 hours, the VCD remained constant at approximately 59 million cells / mL of ECS volume.

[0114] Continued exponential growth of cells over 96 hours indicates that, despite the ambient oxygen sensor readings, cell growth was not oxygen limited up to hour 120. The oxygen transfer limit of the bioreactor under these operating parameters appears to have been reached at approximately 59 million cells / mL.

[0115] The fresh liquid medium had a glucose concentration of 30 mmol / L. The glucose concentration measured by ECS dropped from approximately 30 mmol / L to undetectable amounts after approximately 72 hours. However, glucose consumption remained above approximately 1.5 pmol / cell-day throughout the run. After 72 hours, the continued glucose consumption, combined with continued cell growth, indicates that cell growth was not inhibited by glucose deprivation.

[0116] The oxygen concentration in the liquid medium, measured between the medium bag and the bioreactor, did not substantially decrease below its initial value of 21% solubility during the first approximately 96 hours of operation. Thereafter, the oxygen concentration in the liquid medium gradually decreased, reaching approximately 17% solubility by the end of the run. This suggests that some oxygen was transferred from the perfusion system to the ECS, but not in amounts that supported the density of cells grown in this run.

[0117] Example 13 The reactor according to Example 3 was provided with two sampling ports on the head plate. One sampling port was near the center (axis of rotation) of the reactor and one sampling port was near the periphery of the head plate. The reactor was rotated back and forth at 5 rpm with a 180 degree movement. The reactor was used to grow CHO cells to a final density of 27 million cells / mL. Samples were taken at 21 intervals during the growth period. The average difference in VCD readings between the two sampling locations was 0.9 million cells / mL.

[0118] Two reactors according to Example 4 were provided with two sampling ports. One sampling port was located on the head plate. The other sampling port was located between the two elements furthest from the head plate. One reactor was rotated back and forth at 5 rpm with a 360-degree movement. This reactor was used to grow CHO cells to a final density of 21 million cells / mL. Samples were taken at seven intervals during the growth period. The average difference in VCD readings between the two sampling locations was 1.4 million cells / mL. The second reactor was rotated back and forth at 11 rpm with a 180-degree movement. This reactor was used to grow CHO cells to a final density of 36 million cells / mL. Samples were taken at seven intervals during the growth period. The average difference in VCD readings between the two sampling locations was 1.4 million cells / mL. The difference in VCD readings for the reactor of Example 4 was approximately 8% between the two sampling locations for all readings with an average VCD above 10 million cells / mL.

[0119] A reactor constructed according to Example 6 was provided with one sample port on the inner portion of the head plate and two sample ports on the side of the reactor, one located at element number 5 and the other at element number 9, the element farthest from the head plate. The reactor was used to grow CHO cells and rotated 180 degrees back and forth at 5 rpm. The final cell density was 35 million cells / mL. Samples were drawn from the three sample ports along the culture. The maximum absolute difference in VCD was 2.6 million cells / mL, corresponding to 8.1% of the average reading.

[0120] As shown by the above results, cell density was generally uniform within the reactor both radially and axially.

[0121] Example 14 CHO cells were grown in suspension using a bioreactor according to Example 1. A controller connected to a dissolved oxygen sensor was programmed to maintain an oxygen level in the ECS of 12.5%. The bioreactor was rotated 180 degrees back and forth at 11 rpm. The ramp-up and ramp-down times between full speed (11 rpm) and no speed were 0.2 seconds. There was no pause between bioreactor direction changes (0 second pause delay).

[0122] The bioreactor was operated according to a feed-and-bleed regime similar to Example 12. Secondary medium addition and removal began after 48 hours of operation at 200 mL / day, and this rate increased with VCD. A total of 2.5 L of secondary medium was used. The run lasted approximately 160 hours. The oxygen concentration was maintained at the set point (12.5%) until the end of the run (in contrast to Example 10). The total gas flow was approximately 125 mL / min throughout the run. The gas composition varied from 95% ambient air and 5% carbon dioxide to a mixture of 75% oxygen and 25% ambient air over a period of approximately 30 hours to approximately 140 hours.

[0123] The VCD reached a peak of approximately 70 million cells / mL at approximately 144 hours and remained above 60 million cells / mL until the end of the run. The ability to grow cells to a higher VCD while maintaining the oxygen set point for the reactor of Example 2 is believed to be a result of the additional layer of oxygen transport membrane.

[0124] Another bioreactor according to Example 1 was used to grow CHO cells in suspension. A controller connected to a dissolved oxygen sensor was programmed to maintain an oxygen level in the ECS of 12.5%. The bioreactor was rotated 180 degrees back and forth at 15 rpm. The ramp-up and ramp-down times between full speed (15 rpm) and no speed were 0.2 seconds. There was no pause between bioreactor direction changes (0 second pause delay).

[0125] The bioreactor was operated according to a feed-and-bleed regime similar to Example 12. Addition and removal of secondary medium began at 200 mL / day after 48 hours of operation, and this rate increased with VCD. A total of 5 L of secondary medium was used. The run lasted approximately 192 hours. The oxygen concentration was maintained at the set point (12.5%) until a VCD of 75 million cells / mL was reached. The total gas flow began at 100 mL / min and increased to 150 mL at approximately 126 hours of culture. The gas composition was varied from 95% ambient air and 5% carbon dioxide to a mixture of 80% oxygen and 20% ambient air over a period of approximately 36 hours to approximately 144 hours.

[0126] The VCD reached a peak of about 103 million cells / mL at about 168 hours and remained above 100 million cells / mL until the end of the run. The ability to grow cells to a higher VCD for the reactor of Example 2 while maintaining the oxygen set point for the higher VCD is believed to be a result of the additional layer of oxygen transport membrane.

[0127] Example 15 CHO cells were grown in suspension using a bioreactor according to Example 5. Approximately 15.5 L of first medium was fed in batch mode, consisting of 5.5 L in ECS and 10 L in a bottle in a recirculation loop connected to a perfusion membrane. The bioreactor was rotated 180 degrees back and forth. The ramp-up and ramp-down times between full speed and no speed were 0.2 seconds. There was no pause between bioreactor direction changes (0 second pause delay). The bioreactor was rotated at 5 rpm during the first part of the run and 7 rpm during the second part of the run.

[0128] Glucose was substantially depleted after approximately 70 hours of operation. At this point, the VCD was approximately 25 million cells / mL. Cell growth continued until the end of the run on lactate or hybrid metabolism. The final VCD was approximately 32 million cells / mL.

[0129] Gas was fed in parallel to the elements of the composite reactor. The total gas flow was 700 mL / min. The oxygen concentration measured at the edge of the bioreactor was maintained at the set point (12.5%) by varying the oxygen concentration of the gas until approximately the end of the 96-hour run. The oxygen concentration measured on the head plate near the center of the bioreactor decreased from near the set point at about 24 hours to near zero at about 72 hours. During this time (72 hours), the rotation speed was increased from 5 rpm to 7 rpm (linear velocity 1.2 * 10 -2 m / s). With the change in velocity, the oxygen concentration measured on the head plate near the center of the bioreactor increased to approximately 5% and remained at this level until the end of the run. The kLa measured at approximately 80 hours was 17.2 h -1 The gas mixture delivered at this point was approximately 60% oxygen and 40% air.

[0130] VCD was measured at various times throughout the run at four fittings located at the edge of the head plate, in the center of the head plate, between elements in the center of the reactor, and within the element furthest from the head plate. VCD measurements were similar, i.e., had a variance of less than 2.5 million cells / mL between all four fittings throughout the run. Metabolite measurements were also similar throughout the run between the four sampling locations.

[0131] Example 16 CHO cells were grown in suspension using a bioreactor according to Example 6. Approximately 30 L of first medium was fed in batch mode, consisting of 10 L in the ECS and 20 L in a bottle in a recirculation loop connected to a perfusion membrane. The bioreactor was rotated 180 degrees back and forth. The ramp-up and ramp-down times between full speed and no speed were 0.2 seconds. There was no pause between bioreactor direction changes (0 second pause delay). The bioreactor was rotated at 5 rpm during the first part of the run and 7 rpm during the second part of the run.

[0132] Glucose was substantially depleted after approximately 70 hours of operation. At this point, the VCD was approximately 32 million cells / mL. Cell growth continued until the end of the run on lactate or hybrid metabolism. The final VCD was approximately 35 million cells / mL.

[0133] Gas was fed in parallel to the elements of the composite reactor. The total gas flow was 700 mL / min. The oxygen concentration measured at the edge of the bioreactor was maintained at the set point (12.5%) by varying the oxygen concentration of the gas until approximately 64 hours into the run. After 64 hours, the oxygen began to drop below the set point. This is likely due to the higher viable cell density and more active cell metabolism at this time compared to the other bioreactor runs. The oxygen concentration measured on the head plate near the center of the bioreactor decreased from near the set point at approximately 24 hours to near zero at approximately 64 hours. At 77 hours, the rotation speed was increased from 5 rpm to 7 rpm (linear velocity 1.2 * 10 -2 m / s). With the change in speed, the oxygen concentration measured at the edge of the bioreactor increased, returned to the set point, and remained there until the end of the run. Similarly, the oxygen concentration measured on the head plate near the center of the bioreactor increased to about 3% and then steadily increased until the end of the run. The kLa (5 rpm rotation speed) measured at about 96 hours was 10.2 h at the edge of the bioreactor. -1 , and 12.0 h near the center of the bioreactor. -1The gas mixture delivered at this point was approximately 60% oxygen and 40% air.

[0134] VCD was measured at various times throughout the run at three fittings located at the edge of the head plate, between elements in the center of the reactor, and within the element furthest from the head plate. VCD measurements were similar, i.e., had a variance of less than 3.5 million cells / mL throughout the run, among all three fittings. Metabolite measurements were also similar throughout the run among the four sampling locations.

[0135] Example 17 HEK293 cells were grown using a bioreactor constructed as described in Example 7. The bioreactor was rotated 180 degrees back and forth at 11 rpm. The bioreactor was operated according to a feed-and-bleed regime similar to that described in Example 12. The feed-and-bleed regime was initiated after 48 hours of culture at 100 mL / day and gradually increased to 1200 mL / day at 120 hours of culture. HEK293 cells expanded to a final VCD of 75 million cells / mL with a doubling time of 28.9 hours. Specific glucose consumption observed during the feed phase was 4 pmol / cell-day. The dissolved oxygen concentration reached a set point of 12.5% ​​solubility over the 48-hour culture period and was controlled at the set point by the addition of pure oxygen to the gas stream. At approximately 132 hours of culture, at a VCD of 40 million cells / mL, the percentage of oxygen in the gas stream reached its maximum of 80%, and dissolved oxygen dropped from a set point of 12.5% ​​to undetectable levels. The inability to maintain oxygen at the set point for VCDs higher than 40 million cells / mL is believed to be related to a higher glucose consumption rate compared to the bioreactor and process described in Example 12.

[0136] The above description provides several examples of reactors, parts of reactors, methods of making reactors, and systems and processes for growing cells in reactors. Any one or more aspects of one example can be combined with any one or more aspects of one or more other examples to provide further examples of reactors, parts of reactors, methods of making reactors, and systems and processes for growing cells in reactors.

Claims

1. 1. A method for growing cells in suspension, comprising providing a bioreactor with one or more membranes, optionally gas transfer membranes, and moving liquid medium in the extracapillary space of said bioreactor against said membranes.

2. The method of claim 1 , wherein the relative movement is provided by movement of the bioreactor.

3. 3. The method of claim 1 or 2, wherein the bioreactor has a longitudinal axis, and optionally the bioreactor provides a generally cylindrical plenum for growing cells, the longitudinal axis being a cylindrical axis, and the membranes comprising hollow fiber membranes extending in multiple directions perpendicular or oblique to the longitudinal axis.

4. 4. The method of claim 3, wherein the bioreactor is generally cylindrical and the longitudinal axis is a cylindrical axis, or the bioreactor includes a potting head, the potting head having an inner surface that forms a segment of a circle around the longitudinal axis.

5. The method of any one of claims 2 to 4, wherein moving the bioreactor comprises rotating the bioreactor in clockwise and counterclockwise directions about an axis of rotation passing through the bioreactor.

6. 6. The method of claim 5, wherein the axis of rotation is the longitudinal axis of the bioreactor.

7. 7. The method of claim 5 or 6, wherein the axis of rotation is a) horizontal, b) passes through the center of the bioreactor, and / or c) perpendicular to the membrane.

8. 8. The method of any of claims 1 to 7, wherein the bioreactor is rotated in a pattern that includes 5 or more changes of direction and / or decelerations and accelerations (optionally stopping and restarting) in one direction per minute.

9. 9. The method of any of claims 1-8, wherein the bioreactor is rotated less than 360 degrees in one direction and then less than 360 degrees in the opposite direction in a repeating pattern.

10. The method according to any of claims 1 to 9, wherein the bioreactor is rotated at a rotation speed in the range of 1 to 25 or 2 to 15 rpm.

11. 11. The method of any of claims 1 to 10, wherein the bioreactor is moved according to any of the movements described herein or similar movements adjusted for different size reactors.

12. The method of any of claims 1 to 11, wherein the bioreactor is inverted and re-inverted.

13. the membranes are hollow fiber membranes, the membranes comprise hollow fiber gas transport membranes, the membranes have an outer diameter of 0.3 mm or more, the membranes are arranged in layers with spaces between adjacent layers, the membranes are stacked with a spacing of at least 0.2 mm, the membrane packing density is 25% or less, or 20% or less, or 15% or less, or in the range of 8-12%, or the volume of the extracapillary space of the bioreactor is 1 mm or less of the surface area of ​​the gas transport membrane. ２ 1-20, 2-20, or 2-10 mm per ３ / mm ２ The method according to any one of claims 1 to 12, wherein the range is

14. 14. The method of any of claims 1-13, wherein the bioreactor operates substantially without a continuous gas phase or headspace, and optionally, less than 5%, or 4%, or 3%, or 2%, or 1% of the volume of the ECS is filled with a continuous gas phase.

15. 15. The method of any of claims 1 to 14, further comprising transferring cells from said bioreactor to a second bioreactor, said second bioreactor being operated according to the method of any of claims 1 to 14.

16. 16. The method of claim 15, wherein the second bioreactor is larger than the bioreactor.

17. 17. The method of claim 16, wherein the bioreactor is rotated at a first rotational speed and the second bioreactor is rotated at a second rotational speed, the second rotational speed having a lower speed measured in revolutions per minute than the first rotational speed.

18. Optionally, a method of growing cells in a bioreactor comprising moving liquid medium within a cell growth area of ​​the bioreactor by moving the bioreactor in a repeating pattern having a cycle time of 12 seconds or less.

19. 20. The method of claim 18, wherein the bioreactor has a longitudinal axis and hollow fiber membranes extending in multiple directions perpendicular or oblique to the longitudinal axis, and moving the bioreactor comprises rotating the bioreactor about the longitudinal axis, and the hollow fiber membranes comprise gas transport membranes.

20. 1. A method of growing cells in a bioreactor comprising applying power and, optionally, moving the bioreactor, thereby moving a liquid medium in a cell growth area of ​​the bioreactor, wherein the applied power exceeds the average power for a material portion, i.e., for 20% or more, 25% or more, or 33% or more, optionally up to 50% of the movement cycle.

21. 21. The method of claim 20, wherein the bioreactor has a longitudinal axis and hollow fiber membranes extending in multiple directions perpendicular or oblique to the longitudinal axis, and moving the bioreactor comprises rotating the bioreactor about the longitudinal axis, and the hollow fiber membranes comprise gas transport membranes.

22. A bioreactor having an axis and hollow fiber membranes extending in multiple directions perpendicular or oblique to the axis, the hollow fiber membranes including gas transport membranes; a stand for supporting the bioreactor with the axis of the bioreactor generally horizontal; and a power unit configured to rotate the bioreactor about the axis; Including, the system.

23. 23. The system of claim 22, wherein the bioreactor provides a plenum for growing cells, the plenum for growing cells having an interior surface that defines a segment of a circle about the axis.

24. 24. The system of claim 23, wherein the inner surface is provided by a potting head that holds the ends of the hollow fiber membranes.

25. 25. The system of claim 23 or 24, wherein the plenum is generally cylindrical and the axis is a longitudinal axis or cylindrical axis.

26. A system according to any one of claims 22 to 25, wherein the power unit comprises a motor or actuator, for example an electric, hydraulic or pneumatic actuator.

27. 27. The system of any of claims 22 to 26, comprising a controller programmed or otherwise configured to generate a pattern of movement of the bioreactor via the power unit.

28. 28. The system of any of claims 22-27, wherein the pattern or movement includes one or more of: a) rotating the bioreactor about the axis in a clockwise and counterclockwise direction; b) moving the bioreactor in a repeating pattern having a cycle time of 12 seconds or less; c) rotating the bioreactor in a pattern that includes 5 or more changes of direction and / or decelerations and accelerations (optionally stopping and restarting) in one direction per minute; d) rotating the bioreactor less than 360 degrees in one direction and then less than 360 degrees in the opposite direction in a repeating pattern; e) rotating the bioreactor at a rotational speed ranging from 1 to 25 or 2 to 15 rpm; f) moving the bioreactor according to any of the movements described herein or similar movements adjusted for different size reactors; and g) the bioreactor is inverted and re-inverted.

29. 29. The system of any of claims 22-28, wherein the bioreactor comprises a plurality of elements, each element comprising a set of membranes potted separately from the membranes in the other elements.

30. The system of any one of claims 22 to 29, wherein the membrane has an outer diameter of 0.3 mm or greater.

31. The system according to any one of claims 22 to 30, wherein the membranes are arranged in layers with spaces between adjacent layers.

32. A system according to any one of claims 22 to 31, wherein the membranes are stacked with a spacing of at least 0.2 mm.

33. The system of any of claims 22 to 32, wherein the membrane packing density is 25% or less, or 20% or less, or 15% or less, or in the range of 8-12%.

34. The volume of the extracapillary space of the bioreactor is 1 mm2 of the surface area of ​​the gas transport membrane. ２ 1-20, 2-20, or 2-10 mm per ３ The system according to any one of claims 22 to 33, wherein the range is

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