Bioreactor

Abstract

A bioreactor for culturing cells is described. A screen suitable as a cell growth scaffold may comprise cross fibers. The screen may be loosely accommodated in a screen holder, which in turn may be contained within a manifold assembly. The lower manifold, the screen holder, and the upper manifold may have the same or similar internal opening profiles. The flow of the liquid culture medium may occur upward through the screen array and then flow over a weir in the presence of air pockets, into a trench and a pump. The screen holder may have slits, the outer-facing junctions of which are circular, and may have grooves, the inner-facing edges of which are circular. These components may be located within an incubator suitable for maintaining the required environmental conditions and cleanliness.

Description

[0001] This application is a divisional application with application number 201780052344.9, application date August 25, 2017, and invention name “Bioreactor”.

[0002] This patent application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 380,414, filed on August 27, 2016, which is incorporated herein by reference in its entirety. Technical Field

[0003] Embodiments of the present invention relate to bioreactors. Background Art

[0004] Bioreactors are used to expand cell populations, such as stem cells or other anchorage-dependent cells. However, there is still a need for improvement, for example with respect to ease of use, automation, and reproducibility of procedures. There is also a need for cultures to produce large numbers of cells (e.g., billions of cells if possible). Summary of the invention

[0005] In one embodiment of the present invention, a bioreactor may be provided, comprising: a storage container for containing a liquid culture medium; a conduit, from the lower end of the conduit container to the upper end of the conduit, providing a flow path in a generally vertical direction upward from the storage container; a culture structure located in the conduit, the top of the conduit being located at a position higher than the top of the culture structure; and a circulation system for allowing the liquid culture medium to flow upward through the conduit through or past the culture structure, wherein the upper end of the conduit comprises an overflow wall surrounded by a moat on its outer side, the height of the moat being lower than the top of the overflow wall, the upper end of the conduit and the moat being in contact with a pocket region, the pocket region being defined by a structure connected to the conduit, and the pocket region being in fluid communication and isolation from the outside of the pocket region. When the circulation system is in operation, the liquid culture medium flows on the overflow wall in the pocket region, and the liquid culture medium contacts the gas contained in the pocket region, and the liquid culture medium overflows into the moat and is removed from the moat by the circulation system.

[0006] In one embodiment of the present invention, a bioreactor may be provided, comprising: a storage container for containing liquid culture medium; a manifold assembly, the manifold assembly comprising an upper manifold and a lower manifold, the lower manifold having a lower end extending into the storage container; a screen support, the screen support holding a plurality of screens suitable as a cell support; and a circulation system for flowing the liquid culture medium through the manifold assembly and through the screen support, wherein the manifold assembly and the screen support provide a flow path through the lower manifold and the screen support and the upper manifold, wherein the screen support is contained within the manifold assembly, and wherein the screen is contained within the screen support but has at least some ability to move relative to the screen support in at least one direction.

[0007] In one embodiment of the present invention, a bioreactor may be provided, comprising: a storage container for containing liquid culture medium; a manifold assembly, the manifold assembly comprising an upper manifold and a lower manifold, the lower manifold having a lower end extending into the storage container; a screen support, the screen support holding a plurality of screens suitable for use as a cell support; the screen support may be contained within the manifold assembly; a circulation system for allowing liquid culture medium to flow through the manifold assembly and through the screen support, wherein the manifold assembly and the screen support provide a flow path through the lower manifold and the screen support and the upper manifold, wherein the screen support has grooves on its inner surface and has slits through its outer surface, and wherein the grooves and slits are configured so that the screen can be inserted through the slits and supported by at least one of the grooves and the slits.

[0008] In one embodiment of the present invention, a screen may be provided, wherein at least some of the screens include a plurality of fibers, and within a single screen, the fibers are sequentially arranged in at least first, second, third and fourth layers, the fibers within each layer being generally parallel to each other, wherein the fibers in the first layer are generally parallel to the fibers in the third layer, and the fibers in the second layer are generally parallel to the fibers in the fourth layer, and when viewed perpendicular to the plane of the screen, the fibers in the third layer are not aligned with the fibers in the first layer, and the fibers in the fourth layer are not aligned with the fibers in the second layer.

[0009] In one embodiment of the invention, a bioreactor can be provided with a rotor that can assume more than one angular orientation during a cell growth protocol. The rotor can hold a screen on which the cells are grown. Depending on the orientation of the rotor and the screen, liquid culture medium can be caused to flow through the screen or approximately parallel to the screen.

[0010] In one embodiment of the invention, a bioreactor can be provided with a storage container having features at the bottom that help to use as much liquid culture medium as possible. These features can include a sump near where the pipe draws fluid upward from the storage container, and a rotating rod that can push the liquid culture medium toward the pipe. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Embodiments of the present invention are further described but are by no means limited to the following description.

[0012] Figure 1A The general layout of some major components of the bioreactor according to an embodiment of the present invention is shown.

[0013] Figure 1B Shown in cross section Figure 1A parts.

[0014] Figure 2A The assembly is shown in cross section and includes a lower manifold and an upper manifold.

[0015] Figure 2B Shown with Figure 2A The same assembly with the upper manifold highlighted.

[0016] Figure 2C Shown with Figure 2A The same assembly with the lower manifold highlighted.

[0017] Figure 3A A three-dimensional view of the screen holder is shown without the screen.

[0018] Figure 3B It shows Figure 3A Exploded 3D view of the screen support.

[0019] Figure 3C is a three-dimensional view showing a screen support with a screen therein. For clarity of illustration, one portion of the screen support is a different color than another portion.

[0020] Figure 3D It is shown Figure 3C 3D view of a screen holder with a portion of the screen holder missing to better show the screen.

[0021] Figure 3E It is shown Figure 3D 3D view of the screen holder with some screens missing.

[0022] Figure 4A is a view of the closure of the screen support showing the circular entrance to the slot in the screen support.

[0023] Figure 4B Another view of the closure of the screen support from a different perspective.

[0024] Figure 4C is a view of a partial area of ​​a closure.

[0025] Figure 5A A screen fabricated according to an embodiment of the present invention is shown.

[0026] Figure 5B A close up view of a similar screen is shown.

[0027] Figure 5C The spacing parameters of the screens are shown, e.g. Figure 5A and 5B The screen in the.

[0028] Figure 5D The placement of the fibers within the screen and their interlacing are shown in more detail.

[0029] Fig. 6A Possible leakage flow paths are shown.

[0030] Figure 6B Shown are locations where liquid culture medium is present during typical operation of the system.

[0031] Fig. 7A Three panels are shown as part of a screen support to illustrate the locating slots.

[0032] Figure 7B It is shown Fig. 7A A cross-sectional view of the three-sided parts and the pusher attachment.

[0033] Figure 7C A pusher attachment is shown.

[0034] Fig.7D The recesses and bosses for engaging the screen support with the upper manifold are shown.

[0035] Figure 8 Details of the seal are shown.

[0036] Fig. 9 The figure shows the placement of two bioreactors according to an embodiment of the present invention in an incubator.

[0037] Fig. 10A Flow paths are shown including various gases and liquids into and out of the incubator and into and out of the storage containers and manifold assembly.

[0038] Fig. 10B Shown is a spray head connected to a storage container.

[0039] Fig.11 The general layout of a bioreactor according to another embodiment of the present invention is shown.

[0040] Fig.12 The general fluid flow paths within the bioreactor are shown.

[0041] Fig.13 A storage container base comprising several recesses is shown.

[0042] Fig.14 The riser and nearby components are shown.

[0043] Fig.15 An exploded view of the rotor chamber and rotor is shown.

[0044] Figures 16A-16C Various views of the rotor are shown.

[0045] Fig.17 Details related to the surface level of the liquid in the bioreactor are shown.

[0046] Fig.18 The packing structure inside the riser is shown.

[0047] Fig.19A Results of computational fluid dynamics modeling of the entire system at a first liquid flow rate are shown.

[0048] Fig.19B Results of computational fluid dynamics modeling of the entire system at a second liquid flow rate are shown.

[0049] Fig.19C Results of computational fluid dynamics modeling of the entire system at a third liquid flow rate are shown.

[0050] Fig. 20 Results of computational fluid dynamics modeling near each screen are shown. DETAILED DESCRIPTION

[0051] An important phenomenon in cell culture is the diffusion distance limitation of nutrients and waste products during the growth and maintenance of cells and tissues. Tissues within organisms organize themselves so that cells are never separated from blood vessels or transport pathways by more than a certain distance. The maximum distance that cells can be away from a transport container such as a blood vessel is on the order of a few hundred microns. An important consideration in the design of a bioreactor is to provide a geometry in which cells are never separated from the liquid culture medium by a distance greater than a few hundred microns.

[0052] Another phenomenon related to isolated cells surrounded by liquid is the movement of cells through the liquid. The physical properties of the liquid medium (density, viscosity) are generally similar to those of water. Cells loose in a liquid medium are slightly denser than the liquid medium surrounding them, which causes the cells to fall or sink under the influence of gravity. If a cell is considered to be a sphere moving in a viscous liquid, then Stokes' law describes this situation. In this case, the sedimentation rate is given by

[0053] V=(ρcell-ρfluid)*g*D^2 / (18*μ)

[0054] Where D is the diameter of the sphere, (ρcell-ρfluid) is the difference in density between the sphere and the fluid, g is the gravitational acceleration, and μ is the viscosity of the fluid. This equation applies to laminar flow conditions.

[0055] Another consideration in cell culture is to provide a gaseous atmosphere in the incubator. This results in dissolved gases entering the cell culture medium, containing sufficient oxygen and carbon dioxide at the desired concentrations of about 20% and 5%, respectively, and having the desired relative humidity (e.g., about 95%). Another consideration is to provide a desired temperature, such as approximately normal human body temperature, such as 37.0°C.

[0056] Another consideration in cell culture is the shear stress generated by the liquid that may flow through the cells attached to or being attached to the substrate or cell culture support. It is hoped that this shear stress does not exceed a certain value and does not cause the cells to break away from the culture medium or support. When the cells are still in the process of forming an attachment to the substrate or support, it is believed that small shear stress is particularly important in the early stages of cell inoculation in a bioreactor. For mesenchymal stem cells, it is believed that it is desirable to keep the local shear stress below 0.1 Pa.

[0057] Herein, a first embodiment of the invention is described in which cells are statically inoculated onto a support in the form of a mesh, which is then mounted within a bioreactor, wherein liquid culture medium is perfused through the mesh approximately perpendicular to the mesh.

[0058] Reference now Figure 1A and 1B , shows a bioreactor 10 according to an embodiment of the present invention. The bioreactor 10 may include a storage container 60 suitable for containing a desired amount of liquid culture medium. The bioreactor 10 may also include a storage container cover 62 corresponding to the storage container 60. The storage container cover 62 may be connected to the storage container 60 by snaps or other suitable fasteners or accessories. The storage container cover 62 may have an opening 64 therethrough, the opening 64 being suitable for receiving a flow structure extending through the opening 64 and extending below the surface of the liquid culture medium in the storage container 60. As shown, the flow structure may include various components assembled together and may receive or surround a screen support 200. The screen support 200 may in turn hold a screen 300 on which cells reside and grow. The screen array 300 may be referred to as a culture structure.

[0059] Upper and lower manifolds

[0060] More specifically refer to Figure 2A , 2B 2C, the flow structure may include a manifold assembly including a lower manifold 82 and an upper manifold 86. The lower manifold 82 and the upper manifold 86 may be mated together and in combination may surround and enclose the screen support 200. The lower manifold 82 may extend downwardly sufficiently into the storage container 60 so that the bottom end of the lower manifold 82 may extend below the desired level of liquid culture medium stored in the storage container 60.

[0061] The interior of the lower manifold 82 may be a generally tight fit relative to the exterior of the screen support 200. The lower manifold 82 may have a flange 84. The screen support 200 may rest on the flange 84, but the screen support may also be able to move slightly upward in the vertical direction.

[0062] Upper manifold 86 may be adapted to mate with lower manifold 82. Upper manifold 86 may define an interior passage therethrough that is in fluid communication with the interior of lower manifold 82 and, if screen support 200 is within lower manifold 82, the interior of screen support 200. Upper manifold 86 may have an interior that is similar in shape to the interior of lower manifold 82. The interior of upper manifold 86 and the interior of screen support 200 may have similar or identical shapes and dimensions such that there is little or no change in cross-sectional flow area and change in flow direction as flow passes through lower manifold 82, then through screen support 200, and then through upper manifold 86.

[0063] The upper manifold 86 may have an upper end above the location where it joins the lower manifold 82, which is a weir or overflow wall 112. The weir 112 may be a generally horizontal edge that defines the perimeter of a passage through the upper manifold 86. The weir 112 may define the liquid level within the upper manifold 86 and associated components when liquid is present and flowing in a system having air space in the upper portion of the upper manifold 86. The exterior of the weir 112 may be a moat 116, the bottom of which may be lower than the top of the weir 112. The moat 116 may be adapted to receive and contain liquid that overflows the weir 112. The moat 116 may further have a sump 118, which may be a localized depression that is smaller than the moat 116 and lower in height than other portions of the moat 116. An outlet channel may also be provided in fluid communication with the sump 118, through which liquid culture medium may exit to other fluid circuit portions.

[0064] The upper manifold 86 can be engaged with the lower manifold 82 in a slidable manner, and the slidable manner is guided by the feature of the upper manifold 86, and the feature of the upper manifold 86 is parallel, similar in shape and has similar size relative to the corresponding feature of the lower manifold 82, but allows enough movement to be loose. As shown in the figure, relative to the feature of the lower manifold 82, the feature of the upper manifold 86 is positioned outward. The upper manifold 86 can have a contact with the lower manifold 82, and the lower manifold 82 limits the movement of the upper manifold 86 towards the lower manifold 82, forming a stop constraint. Or, when the appropriate surface of the upper manifold 86 (which can be flat) contacts the upper surface of the screen support 200, and the lower surface of the screen support 200 contacts the appropriate surface of the lower manifold 82 (which can be flat), the movement of the upper manifold 86 and the lower manifold 82 towards each other can be stopped.

[0065] A top cover 130 may also be provided to cover the top of the upper manifold 86. The top cover 130 may be at least approximately a flat plate. If desired, the top cover 130 may have reinforcing ribs. The top cover 130 may be permanently connected to the upper manifold 86, and a seal may also be provided between the top cover 130 and the upper manifold 86. If desired, some form of fastener connecting the top cover 130 and the upper manifold 86 may also be provided.

[0066] As shown, the opening 64 through the storage container cover 62 can have a raised edge or lip 68 around it, and the lower manifold 82 can have a flange 83 around the lower manifold 82 on its exterior. The flange 83 can rest on the raised edge or lip 68 to define the spatial relationship between the lower manifold 82 and the storage container cover 62. The raised edge or lip 68 can be provided to prevent any condensation of liquid water from dripping into the storage container 60 if it occurs on the storage container cover 62, and if such condensation is not sterile. Such condensation can be confined to the top (exterior) surface of the storage container cover 62. Various designs of such a lip are possible.

[0067] Seals and clamps

[0068] The lower manifold 82 may have a lower manifold sealing groove 92 adapted to accommodate a compressible seal 94 for forming a seal between the lower manifold 82 and the upper manifold 86. The compressible seal 94 is shown as a rectangular cross-sectional shape with deformable triangular fingers on one surface. Of course, other cross-sectional shapes are also possible. The preload between the upper manifold 86 and the lower manifold 82 can be generated by a clamping device, which can be an eccentric type clamping device. Two such clamping devices 97A, 97B are shown. When the described stop condition is reached based on the position of the upper manifold 86 and the lower manifold 82 relative to each other, the compressible seal 94 can occupy a compressed configuration.

[0069] The upper manifold 86 may have an upper manifold sealing groove 96 adapted to hold a compressible sealing member 98 to form a seal between the upper manifold 86 and the top cover 130. The compressible sealing member 98 is shown as a typical O-ring. The preload between the top cover 130 and the upper manifold 86 may be created by snaps (not shown), clamps (not shown), or any other suitable design features. Another possibility is that the top cover 130 may be permanently connected to the upper manifold 86, for example, by an adhesive.

[0070] Screen support

[0071] In one embodiment of the present invention, the screen 300 can be held by the screen support 200. The screen support 200 is Figures 3A-3E and Figures 4A-4C The screen support 200 can hold a desired number of screens at desired spacing positions and orientations. Figures 3A-3E and Figures 4A-4C The screen holder 200 shown in FIG. 1 holds 15 screens 300. It may be desirable to have a number of screens no greater than about 15 to avoid creating oxygen concentration gradients in the liquid culture medium during operation of the bioreactor.

[0072] Typically, the screen support 200 can surround the hollow interior space and can be any of a variety of shapes, such as circular, rectangular, etc. As shown, the screen support 200 has a general shape of a rounded rectangle (both inside and outside thereof). The screen 300 held by the illustrated screen support 200 is rectangular. For ease of description, the screen support 200 is described here using directional indications such as the front and back, the side, and the horizontal and vertical, which generally correspond to the orientation or position shown in the assembled bioreactor 10. However, it should be understood that these directional designations are somewhat arbitrary.

[0073] When the screen 300 is in place in the screen support 200, there may be a desired amount of space in the vertical direction between the screen 300 and its nearest neighboring screen. In the bioreactor 10 shown in the figure, the flow of liquid culture medium can generally flow perpendicular to the flat surface of the screen 300, through the open space in the screen 300.

[0074] The cell culture area may contain, for example, about 15 such screens 300 spaced a sufficient distance from each other so that the screens do not touch each other and some lateral flow of liquid culture medium is possible if desired.

[0075] The screen support 200 can be a simple shape of a hollow, some of which form a complete periphery, and its shape is a shape such as a circle or rectangle (possibly with rounded corners). Generally, the screen support 200 can be made into a single piece, such as by an additive manufacturing process. However, more typically, the screen support 200 can be made of two parts, such as two molded plastic parts, which are connected to each other. It is shown that the screen support 200 is made of two mutually engaged parts. The screen support 200 may include a three-sided piece 220 and a closure 240 that can be engaged with the three-sided piece 220. The three-sided piece 220 may include a first side segment 222A, a rear segment 222B, and a second side segment 222C opposite to the first side segment 222A in sequence. The closure 240 may include at least one section, which can be regarded as a front segment 242B. As shown, the closure 240 may additionally include a first side segment 242A and a second side segment 242C. Therefore, in sequence, there are a first side segment 242A, a front segment 242B and a second side segment 242C. The first side segment 242A can be engaged with the first side segment 222A, and the second side segment 242C can be engaged with the second side segment 222C. However, the closure 240 can also include only the front segment 242B. As shown, the front segment 242B and the rear segment 222B are substantially parallel to each other, and the first side segment 222A and the second side segment 222C are substantially parallel to each other, and the first side segment 242A and the second side segment 242C are substantially parallel to each other. However, other spatial relationships are also possible.

[0076] The engagement feature may include a deformable tab 800, and may also include an opening 224A in the first side segment 222A and a similar opening 224C in the second side segment 222C, the openings 224A and 224C being appropriately designed to engage with the tab 800. The tab 800 may be elastically deformable between an engaged position as shown and a release position in which the deformable tab 800 is bent inwardly enough to produce disengagement. The tab 800 may include a living hinge, and elasticity elsewhere in the closure 240 may also facilitate changes in size or shape to allow or facilitate disengagement.

[0077] Screen support 200 may have upper and lower edges, which may be flat and may be parallel to each other.

[0078] Slots and grooves in the screen support, and rounded edges near the slots and grooves

[0079] The screen support 200 may include various grooves and slots that define the position of the screens 300 and provide mechanical support for the screens 300. As shown, the screen support 200 is capable of holding 15 of the screens 300, but of course other numbers of screens 300 are possible. The spacing distance between the screens 300 can be selected from a combination of considerations such as the fluid flow pattern between the screens 300 and the desired overall packing density of cells in the cell culture area of ​​the bioreactor 10.

[0080] If the screen support 200 is made in one piece (similar to the one shown but with three face pieces 220 and closure piece 240 connected together), the screen 200 can be inserted into or removed from the screen support 200. This can be done from a side direction.

[0081] In the screen support 200 as described and illustrated, when the three-sided piece 220 and the closure piece 240 have been assembled to each other, the screen 300 can be inserted into the screen support 200 or removed from the screen support 200 without disassembling these parts from each other. This can be done from the side direction. As shown, the first side section 222A has a groove 170, and the second side section 222C has a groove 170. The rear section 222B has a groove 170. The front section 242B has a seam, and the screen 300 can pass through the seam without disassembling the screen support 200. The grooves 170 can be substantially coplanar with each other, and can have similar or identical sizes to each other in the vertical direction. The seam 180 can be parallel or coplanar with at least some of the grooves 170. The vertical dimension and height of the seam 180 can be the same as the vertical dimension and height of the groove 170, but it is sufficient if the vertical dimension and height are simply roughly the same as each other. These various features can combine and interact so that when screen support 200 is assembled, screens 300 can be slid into place through slots 180 and supported by grooves 170 and slots 180. The desired spacing or spacing between screens 300 is maintained by the material of screen support 200 near slots 170 and by the material of screen support 200 held between slots 180 (joints).

[0082] As shown in the figure, closure 240 has three sides, wherein two sides 242A, 242C are short.A sidewall of screen support 200 comprises first side segment 222A and first side segment 242A, and similarly, another sidewall comprises second side segment 222C and second side segment 242C.As shown in the figure, even lug 800 comprises the groove continuous with the groove 170 of corresponding adjacent side segment.But, it is to be appreciated that various other designs are also possible.

[0083] As shown, the grooves 170 in the first side segment 222A and the first side segment 242A are substantially sharp edges. The same is true for the grooves 170 in the second side segment 222C and the second side segment 242C. However, it will be appreciated that other geometries are possible.

[0084] At the front of the screen support 200, the spacers or couplings of the remaining material can be provided with rounded edges on the general exterior of the screen support 200. This can help guide the screen 300 into the slot 180 during the initial period. The screen 300 is inserted into the slot 180. The rounded edges can be semi-cylindrical. If any screen 300 is not completely planar, such as slightly warped out of plane, such rounded edges can also help guide the screen 300 into the slot 180.

[0085] At the rear of the screen support 200, the spacers between the individual screens 300 can be such that the circular edge faces the interior of the screen support 200. When the screen 300 is inserted into the screen support 200, such as at the end of the insertion process, this helps to guide the screen 300 to the desired position. The circular edge can be semi-cylindrical. The circular edge can be semi-cylindrical. If any screen 300 is not completely planar, such as slightly bent out of plane, such a circular edge can also help to guide the screen 300 into the groove 170.

[0086] The partitions at the front of the screen support 200 may define a slot through which the screen 300 may pass. The partitions at the rear of the screen support 200 may define a groove through which the screen 300 may enter, but through which the screen 300 may not pass.

[0087] In the case of the rounded edges of the slot 180 and the rounded edges of the groove 170, the semi-cylindrical curvature is only one of various possible curvatures. Rounded corners with some other desired radius are also possible. Such radius can be smaller or larger than the radius of the semi-cylinder. Other shapes of curves are also possible. Curvature changes along the length of the slot 180 or groove 170 are also possible.

[0088] Screen design

[0089] In an embodiment of the present invention, and referring now to Figures 5A-5D , the bioreactor 10 can have an array of screens 300 within the culture area, which serves as a tissue scaffold on which cells (e.g., anchorage-dependent cells) grow. Each screen 300 itself can include multiple layers of fibers 400, the fibers 400 having an orientation perpendicular to the orientation of the fibers in adjacent layers. In a particular embodiment of the invention, the number of layers of such fibers 400 in a single screen 300 can be, for example, four or five or six layers.

[0090] Typical dimensional parameters of such a screen 300 may be a fiber diameter of 150 microns and a fiber spacing of 200 microns (eg, Figure 5C ), which dimension refers to the distance from the edge of one fiber to the nearest edge of the nearest fiber in the same plane. The fibers 400 in one layer may be staggered relative to the fibers 400 parallel to them and located in different layers of the screen 300, or alternatively, need not be staggered. The fibers 400 may be spaced apart from each other by an appropriate distance so that during initial cell seeding, cells are deposited on the fibers 400 and do not contact cells on adjacent fibers 400. However, the spacing of the fibers 400 may be such that after a certain number of cells have proliferated, cells growing on nearby fibers 400 may contact or grow against each other (a condition known as confluence).

[0091] More specifically, the fibers 400 may exist in two mutually perpendicular directions and may be staggered in the two mutually perpendicular directions. Figure 5D Further described in. Each screen may include a plurality of fibers forming a first layer, the first layer having parallel fibers oriented in a first direction, and may include a second layer having a plurality of parallel fibers oriented in a second direction perpendicular to the first direction, the fiber layer in the second layer being connected to the fibers in the first layer. At least some of the screens include a plurality of fibers, and within a single screen, the fibers are arranged in at least the first, second, third and fourth layers in sequence, the fibers in each layer are generally parallel to each other, wherein the fibers in the first layer are generally parallel to the fibers in the third layer, the fibers in the second layer are generally parallel to the fibers in the fourth layer, and wherein when viewed perpendicular to the flat surface of the screen, the fibers in the third layer are not aligned with the fibers in the first layer, and the fibers in the fourth layer are not aligned with the fibers in the second layer. More specifically, for this viewing direction, the fibers may be located in the middle between the misaligned parallel fibers. Such a configuration can help prevent cells from falling through the screen, particularly during initial seeding, while still providing space for the liquid culture medium to occupy and flow through, and providing growth space for the cells as they proliferate.

[0092] Screen 300 can have a certain number of layers, for example four layers, wherein the culture medium for contacting cells can be obtained at the two surfaces of the four-layer construct, and the culture medium can also be obtained in the openings existing between the layers. It is believed that all features of this situation are more similar to the features (called three-dimensional environment) occurring in natural tissues. It is believed that the three-dimensional environment of the embodiment of the present invention is more conducive to cell proliferation and amplification than the two-dimensional environment. However, it is not desired to be limited to this explanation.

[0093] The screen 300 can be formed by programmed deposition of heated polymer filaments, similar to that described in co-owned U.S. Patent No. 8,463,418. The polymer can be a suitable biocompatible polymer, such as polystyrene. The screen 300 described is non-woven. Alternatively, the screen 300 can be woven if desired. The overall shape of the screen 300 can be flat and rectangular.

[0094] In some prior art cell culture techniques such as culture dishes, cell layers grow on a flat surface and experience a substantially two-dimensional environment. Even if there is adequate nutrient supply and waste removal in this case, this two-dimensional environment is essentially different from the environment (three-dimensional environment) in which cells grow naturally.

[0095] In an embodiment of the present invention, cells are inoculated by being attached to each fiber of the screen.The fiber-fiber size of the screen can be large enough so that when the separated cells are initially attached to the fiber, at least some cells are roughly not in contact with other cells.After cell propagation, and several layers of cells may be produced, wherein initially only one layer of cells is attached to the fiber, the outermost cells may still be independent of the next fiber, or some new cells may be other cells that can contact and be attached to other fibers, i.e. some fiber bridging (being called convergence) may occur.In an embodiment of the present invention, the screen can have a specific number of fiber layers, for example four layers.On both surfaces of the four-layer construct, there is a culture medium that can be used for contacting cells.In any case, it is believed that all features of this situation are more similar to the features (three-dimensional environment) occurring in natural tissue.It is believed that the three-dimensional environment of the embodiment of the present invention is more conducive to cell proliferation and amplification than two-dimensional environment.But, it is not desired to be limited to this explanation.

[0096] The cell culture area may contain, for example, about 10 to 15 such scaffolds spaced a sufficient distance from one another so that the screens do not touch one another and liquid can flow between the scaffolds.

[0097] In terms of biological parameters, the screen used with embodiments of the present invention may have an area of ​​about 7000 square millimeters (length dimension * width dimension), and the portion of the screen exposed to the perfusion (excluding the edge in the groove or seam) may have an area of ​​about 6300 square millimeters. The screen can be formed by four layers of fibers, which are alternated in the direction as described elsewhere herein. On such a screen, an initial inoculation of about 800,000 cells can be deposited, so if 12 screens are used, the population of inoculated cells is 9.6 million cells. At the end of the culture, the cell population has expanded to about 250 million cells. For dynamic culture, the flow rate of the liquid culture medium based on the empty space in the screen can be about 2cm / min. It is believed that based on the empty space of the liquid passing through the screen, a flow rate of 1.6mm / sec will produce a maximum shear stress of 0.1Pa at the fiber edge in the screen, and it is believed that for mesenchymal stem cells, the shear stress should be kept below this value.

[0098] Dimensional Considerations Affecting Flow

[0099] In an embodiment of the present invention, the general flow direction of the liquid culture medium may be perpendicular to and pass through the screen 300 , and as shown in the figure, the direction is vertically upward.

[0100] In an embodiment of the present invention, cells are seeded by attaching to a single fiber 400 of a mesh 300. The fiber-to-fiber size of the mesh 300 can be large enough so that when the separated cells initially attach to the fibers 400, they do not substantially touch other cells. After the cells proliferate, and several layers of cells attached to the fibers 400 may be produced, of which only one layer of cells initially attaches to the fibers 400, the outermost cells may still not be in contact with the cells attached to the nearest neighboring fibers 400, or some new cells may contact other cells attached to other fibers 400, i.e., some bridging (convergence) between fibers may occur. Even if convergence does not occur, after other cells grow, the open space for the passage of liquid culture medium through the mesh is less than the open space at the beginning of the culture.

[0101] In an embodiment of the present invention, the screen 300 can be slightly loosely fitted in the groove 170 or the slit 180 of the screen support 200. This allows the screen 300 to be easily inserted into or removed from the groove 170 and the slit 180. For example, the total height of the screen 300 can be 600 microns, and the vertical dimension of the groove 170 or the slit 180 can be 1 mm. This leaves a gap between the screen 300 and the groove 170 or the slit 180 in the vertical direction. In the horizontal direction, in the left and right directions, the size of the screen 300 can be slightly smaller than the horizontal dimension of the base of the groove 170 on the other side of the screen support 200 defined by the base of the groove 170 on one side of the screen support 200. In the horizontal direction, in the front-to-back direction, the position of the screen 300 can be determined by two limiting structures, between which the screen 300 can have a slight degree of slackness. One of these structures can be the inner wall of the lower manifold 82. Another structure can be the inward end of the boss 216. In the front-to-back direction, the screen may be slightly smaller (at least at the location of the boss 216) than the distance between the boss 216 and the opposing inner surface of the lower manifold 82. Alternatively, some features of the screen support 200 may also be involved in determining the position of the screen 300.

[0102] During flow through the screens, the following forces may act on a horizontally oriented screen: the weight of a single screen when submerged in liquid medium; and the drag caused by the flow of liquid medium (which can be approximated as the pressure difference across the screen support divided by the number of screens). This force balance will determine whether the screen is at the lower edge of the groove, or is pushed up and pinned to the upper edge of the groove, or is floating slightly erratically between the other two positions. At least one of these quantities, the pressure drop, may change during the culture process as the screen becomes more crowded with cells.

[0103] At the same time, it is desirable for most of the liquid culture medium to flow through the openings in the screen 300 .

[0104] In the described stack of screens 300 and within the described screen support 200, there are various possible local flow paths for liquid culture medium. In most of the screen 300, there are open spaces between the fibers 400 through which liquid culture medium can flow. Prior to inoculating cells onto the screen 300, the size of the minimum flow area for a particular opening between the fibers 400 can be approximated as the inter-fiber spacing distance multiplied by the inter-fiber spacing distance. Furthermore, prior to inoculating cells, there are possible flow paths at the edges of the screen 300 when the screen is installed into the screen support 200. Such flow paths are Fig. 6A Flow in such a flow path may be affected by local dimensions, such as the inter-fiber spacing distance, and the gap in the horizontal direction between the screen 300 and the screen support 200, and the gap in the vertical direction between the screen 300 and the screen support 200. However, to simplify the discussion, it can be assumed that for one space between the fibers 400, the area of ​​the leakage flow is the inter-fiber spacing distance multiplied by the inter-fiber spacing distance.

[0105] It may be meaningful to compare the desired flow rate to the leakage flow rate, in the form of a ratio of these two quantities. If, for simplicity, the outer dimensions of the screen are assumed to be a square with a side length of L, the number of cells along the perimeter will be 4*L / delta, where delta is the inter-fiber spacing distance. Then, the total flow area of ​​all these leakage paths will be (4*L / delta)*delta^2, or 4*L*delta. In the same case, for the desired flow rate, the total flow area through the screen is L^2*delta^2. The ratio of the desired flow area to the leakage flow area is L^2*delta^2 / (4*L*delta), or L / (4*delta), or 0.25*(L / delta). In order to have the ideal goal of having most of the flow pass through the openings in the screen 300 rather than through the leakage paths, this motivates the screen 300 to have many cells within the length of the side of the assumed square shape. For example, the system may be designed so that the leakage path including the gap between a single one of the screens and the screen support has a cross-sectional flow area that is less than 10% or 2% of the total flow area of ​​all passages through the single screen.

[0106] A further detail is the assumption that over time and as the cell population increases, the open spaces in the main portion of the screen 300 become smaller. It can be assumed that they have a value that can be called delta crowding (deltacrowded). It is then necessary to make assumptions about the open spaces at the edges of the screen 300. It can be assumed that the open spaces at the edges do not change their size, and that their size remains at a size that can be called the delta edge (deltaedge). The above flow area ratio then becomes L^2*deltacrowded^2 / (4*L*deltaedge), or 0.25*L*deltacrowded^2 / deltaedge. This is of course subject to assumptions specifically about the size of the openings at the edges (i.e., deltaedge), but in general as deltacrowded becomes smaller, the ratio of the flow area for the required perfusion flow divided by the flow area for the leakage flow can become less than the ratio for an empty screen 300.

[0107] Another consideration affecting the bioreactor design can be the rigidity of the screen 300 against bending. When the screen 300 is in the bioreactor and supported by the screen support 200, they are subject to gravity (with or without buoyancy effect depending on whether the liquid culture medium is present), and if the liquid culture medium is moving, they are also subject to the effect of flow forces. Any such force may cause the screen 300 to deform out of plane. Ideally, the screen 300 will not deform so much that they contact each other or loosen from the screen support 200. The rigidity of the screen 300 can be characterized by its bending deformation in a simple bending geometry, wherein two opposite edges are supported as simple supports and the other two edges are not supported. For the screen, its size along the bending direction is about 100mm, the lateral size is about 60mm, and the weight of 14.5 grams applied at the center of the span causes a deflection of 3.1mm. The screen can be designed to be bent at least as rigid as the bending just described. When the screen is supported at four edges rather than the two just described, it will deflect less than in the measurement just described.

[0108] Start the program

[0109] In one embodiment of the present invention, the startup procedure can be as described herein. When the system begins to be used, the storage container 60 can contain liquid culture medium up to the desired liquid level. The liquid level can be higher than the height of the bottom of the lower manifold 82, and the amount of liquid contained can be sufficient for the desired operation. The pump 140 can then be operated to draw fluid from the trench 116. The pump 140 can be a positive displacement pump, such as a peristaltic pump, capable of moving liquid or gas or a combination thereof. Initially, the pump will remove air from the area including the upper manifold 86. This removal of air will cause the level of the liquid culture medium to rise in the lower manifold 82, enter the screen support 300, and then enter the upper manifold 86. Eventually the liquid level will reach the weir level, and the liquid will then flow over the weir 112 into the trench 116. The liquid will then flow into the storage tank 118. The pump 140 draws fluid from the storage tank 118. At some point during startup, the pump 140 may draw a mixture of liquid and gas from the storage tank 118, but this is normal. Eventually a steady state will be reached where a substantially constant volume of gas remains in the area above the upper manifold 86 and the moat 116. As long as the pump 140 continues to operate, there will be a flow of liquid from the weir (overflow wall) 112 into the moat 116 and sump 118 and toward the pump 140. It is believed that the level of liquid in the moat 116 will be fairly close to the bottom of the moat 116. This is Figure 6B The liquid Figure 6B Indicated by dashed hatch pattern.

[0110] Pusher attachment and screen positioning slots

[0111] 7, in an embodiment of the present invention, as described herein, the screen support 200 may include a locating slot 390 that may be used to push the screen 300 into a desired position or to define the position of the screen 300. Such a locating slot 390 may be generally perpendicular relative to the overall orientation of the screen support 200 and the bioreactor 10. The locating slot 390 may intersect with other slots and grooves disposed in the screen support 200.

[0112] Pusher 400 can also be provided as an accessory (for use before or after the actual cultivation). Pusher 400 can be used on one side of screen support 200 to push screen 300 into screen support 200 until they contact a stopper such as the base of groove 170. Pusher 400 can also be used on the opposite side of screen support 200 to push screen 300 out of screen support 200, for example when cultivation is completed or when it is desired to remove screen 300. The locating slits 390 on one side of screen support 200 can have the same or similar size and spacing as the locating slits 390 on the other side of screen support 200, which will enable a single pusher 400 to be used for pushing in two directions.

[0113] To facilitate the described pushing, the pusher 400 may have certain dimensional relationships with appropriate features of the screen support 200. The bosses 420 on the pusher 400 may be sized and appropriately spaced relative to each other so that they may fit into the locating slots 390 of the screen support 200. The height of the bosses 420 on the pusher 400 may be large enough so that the screen 300 may be pushed to the desired extent.

[0114] Features that affect the engagement between the screen support and the lower manifold

[0115] It is also possible that interengagement features that constrain or guide the interengagement between the screen support 200 and the lower manifold 82 can be provided. These features can be referred to as keys and keyways. As shown, the screen support 200 has a recess 210, and the lower manifold 82 has a boss 216. Of course, it can also be the opposite, that is, there is a boss on the screen support 200 and a recess on the lower manifold 82. If the screen support 200 has a certain degree of symmetry, such as a rectangular cross-sectional shape, the key and keyway can limit the number of ways in which the screen support 200 and the lower manifold 82 can be assembled together. When the screen support 200 is assembled with the rest of the bioreactor 10, this arrangement can be used to control the positioning of the screen 300.

[0116] There are some dimensional considerations regarding the insertion of screen support 200 into lower manifold 82. For example, the width of boss 216 (in the horizontal direction) may be less than the width of recess 210.

[0117] If desired, when the screen support 200 is inserted into the lower manifold 82, the boss 216 or part of the recess 210, or both, initially engage each other, and a circular feature may be provided at one end thereof (at the top of the lower manifold 82 feature or the bottom of the screen support 200 feature, or both) to guide the initial engagement between the screen support 200 and the lower manifold 82. It may also be a circular feature, which is provided at other locations on the bottom of the screen support 200, or is provided at the top of the lower manifold 82, or both, to guide the initial engagement between the screen support 200 and the lower manifold 82.

[0118] Incubator

[0119] Reference now Fig. 9, the bioreactor 10 may include an incubator 950. The incubator 950 may maintain conditions within itself that are conducive to cell growth. The incubator 950 may maintain a temperature close to a desired value of physiological temperature. The incubator 950 may also maintain an atmosphere having a desired component. The component may have an oxygen concentration of about 20% and a carbon dioxide concentration of about 5%. The incubator 950 may also maintain a desired relative humidity of the atmosphere within the incubator 950. The incubator 950 may have a front door that provides access to the interior of the incubator 950 for installing or removing major components. The incubator 950 may also have penetrations or passes through its various walls. Such passes and penetrations are described here for a specific design, but it will be understood that other such arrangements are also possible.

[0120] There may be a pump 140 for circulating or controlling the flow of liquid culture medium at a location external to the incubator. The pump 140 may be a positive displacement pump, such as a peristaltic pump. There may be appropriate passages or penetrations to allow these liquids to enter and exit the incubator 950 and enter and exit the pump 140. Positioning the pump 140 outside the incubator may serve certain specific purposes. One such purpose is that the electronics in the pump 140 may have limitations on the humidity to which they can be exposed, which may be lower than the relatively humid conditions typically maintained inside the incubator 950. Positioning the pump 140 outside the incubator 950 eliminates this concern. Additionally, if the pump 140 is located inside the incubator 950, it may generate heat when operating, and this heat may affect the temperature control within the incubator 950. Appropriate controls may be provided for the incubator 950, and automation of the pump and related systems may also be provided if desired.

[0121] The components just described may be suitable for placement in a controlled environment chamber, which may be referred to as an incubator 950. The incubator 950 may have controls to maintain a desired temperature, a desired atmosphere composition, a desired oxygen concentration, a desired carbon dioxide concentration, a desired humidity, a desired value of any other environmental characteristic, or any combination of these inside it. The incubator 950 may have a door that allows these components to pass through, and the door may be closable and sealable. The incubator 950 may also have a passage through the incubator wall or boundary, which may be separate from the door. Such a passage may allow liquid to enter and exit the interior of the incubator 950. The incubator 950 may further have a connection through which oxygen or carbon dioxide may be supplied to the interior of the incubator 950. A pump 140 for circulating liquid culture medium may be located outside the incubator 950. The incubator may be suitable for maintaining sterility or at least cleanliness at and near the components (e.g., components of manifolds 82, 86 and storage container 60).

[0122] The outlet fitting may be connected to an overflow moat 116. The outlet fitting may be off the moat so that a portion of the interior open space of the fitting is at or below the floor level of the moat 116. From the outlet fitting, there may be a pipe or similar fluid carrying device to the inlet of the pump 140. The pipe may be capable of directing fluid (liquid or gas) from the moat 116 to the pump. The pump 140 is shown as being located outside the incubator 1950. This pump location is optional, although its advantages are discussed elsewhere herein.

[0123] Downstream of the pump 140, the flow can return to the incubator 950 and the sparger 186. The sparger 186 can be located within the incubator 950 on top of the storage container 60. The sparger 186 can dispense the liquid culture medium in the form of droplets, which fall into the storage container 60. The atmosphere within the incubator 950 can have a desired oxygen concentration and a desired carbon dioxide concentration, and can also be temperature controlled. During the process of the liquid culture medium passing from the sparger 186 through the atmosphere within the storage container 60, the droplets can exchange gases with the atmosphere within the storage container 60, such as absorbing carbon dioxide from the atmosphere within the storage container 60. If desired, the droplets can also be in thermal equilibrium with the atmosphere within the storage container 60. The droplets can then be collected in the liquid region of the storage container 60.

[0124] The fluid flow through the entire flow path may then be repeated.

[0125] Reference is now made to FIG. 10 , which illustrates various connections through the boundary of the incubator 950 and storage container 60. The bioreactor 10 may include a connection for a pump 140 to withdraw liquid culture medium and reintroduce liquid culture medium. This provides for dynamic circulation or perfusion of the liquid culture medium. If the pump 140 is located outside the incubator 950, the liquid culture medium may be withdrawn from the bioreactor 10, pass through the boundary of the incubator 950, pass through the pump 140, pass through the boundary of the incubator 950 again, and re-enter the bioreactor 10. More specifically, the liquid culture medium may be withdrawn from a sump 118 connected to a moat 116 and may re-enter the storage container 60 through a sparger 186. The atmosphere within the incubator 950 and storage container 60 may be managed in several ways. CO may be supplied from a source external to the incubator 950. 2 , and as shown in the figure, CO 2950. Air can enter the interior of the incubator 950 from the outside of the incubator 950 through the channel shown on the left side of the incubator 950. This incoming air can pass through a filter 960, which can be located outside the incubator 950. Thus, the atmosphere inside the incubator 950 can be a mixture of air and carbon dioxide. The gas space of the storage container 60 can have an air extraction connection through which gas from the gas space of the storage container 60 is pulled from the storage container 60, through the channel through the boundary of the incubator 950, and output to the gas flow pump 150. The gas flow pump 150 can be a peristaltic pump, which can be located outside the incubator 950. Gas from the atmosphere inside the incubator 950 enters the storage container 60 through a filter (not shown) on the port 940. Within the storage container 60, some of that gas is entrained by the sparger 186, which causes aeration and exposes the liquid culture medium to CO 2 and oxygen. Cells obtain CO from the liquid medium 2 and oxygen for its metabolism and ATP cycle for energy transfer. The nozzle 186 shown in the figure has a non-uniform hole size distribution so as to form a spray with desired characteristics and distribution. The liquid enters from above the nozzle 186 near the center of the hole array. The size of the holes located more outward is larger than the size of the holes near where the liquid enters.

[0126] As shown, two channels through the left wall of the incubator 950 are used for liquid culture medium to enter and exit the incubator. There is also a channel through the wall of the incubator through which an air flow pump, which may be a peristaltic pump, pulls atmosphere out of the gas space of the storage container 60. There is also a channel through which air from outside the incubator 950 enters the interior of the incubator and passes through the filter 960.

[0127] Embodiments including a rotor and two different flow directions

[0128] In general, there are at least two possible planar support geometries and flows in a bioreactor. The foregoing embodiments involve flow through the screen approximately perpendicular to the plane of the screen. Another possible geometry involves flow of liquid in a direction approximately parallel to the screen. Each direction has advantages and disadvantages.

[0129] In this next embodiment of the invention, the bioreactor 1010 may have Fig.11 The overall components and arrangement shown in .

[0130] The components described can be attached to a base or frame which defines their location and allows the device to be carried as a unit. A source of carbon dioxide can also be provided external to the device, such as a pressurized tank. Similarly, a source of oxygen can be provided if necessary.

[0131] Flow Path

[0132] Reference now Fig.12 , for an embodiment of the present invention, a fluid flow arrangement for a bioreactor 1010 is shown. As shown, the direction of liquid flow through the cell culture support can be in a generally upward direction relative to gravity. The flow can flow in a circular flow path. In one embodiment of the present invention, the liquid culture medium can flow in a circular path from a storage container, upward through the support, through the pump, through the nozzle, and back to the storage container.

[0133] Pump 1140 can be self-priming by initially pumping gas through the pump to draw liquid from the lower storage container upward into the cell culture area. Thereafter, pump 1140 can move liquid when needed and circulate the liquid through the culture area at whatever rate is desired. Pump 1140 can be a positive displacement pump, such as a peristaltic pump.

[0134] The apparatus may include a storage container 1060, which may be located generally at a height lower than the height of various other components of the apparatus. The bottom of the storage container 1060 may be formed at least in part by a storage container base 1400, which is generally flat and horizontal, having a storage container base upper surface 1410. The storage container 1060 may also be defined by a storage container sidewall and a storage container cover. In addition, the storage container base 1400 may have some features that are recessed below the storage container base upper surface 1410.

[0135] The storage container 1060 may have an agitator therein, such as a magnetic stirring bar (not shown), which may be rotated by rotating an externally applied magnetic field. The stirring bar may be denser than the density of the liquid so that the stirring bar may sink due to gravity and rest on the surface of the storage container base, more specifically the bottom surface of the agitator recess 1420. Other agitator arrangements are also possible, such as rotating rods and paddles.

[0136] The stir bar can be rotated at an appropriate rotation rate to suspend the cells in the liquid so that the liquid drawn into the scaffold contains the appropriate cells.

[0137] Storage container base

[0138] Storage container 1060 may be defined in part by storage container base 1400. Fig.13 , the storage container base 400 may be generally flat and level, but may have a pattern of recesses recessed therein.

[0139] Corresponding to the stirring rod, there may be a stirring recess 1420 recessed into the storage container base 1400. The stirring recess 1420 may be larger in its horizontal dimension than the path swept by the stirring rod. At least some of the stirring recesses 1420 may be generally cylindrical and may have a flat and horizontal bottom. If desired, the depth of the stirring recess 1420 may be less than the vertical dimension of the stirring rod.

[0140] A channel recess 1430 may further be provided, which may be substantially straight in the length direction. A storage container recess 1440 may also be provided. The channel recess 1430 may intersect with the agitator recess 1420 and may intersect with the storage container tank recess 1440.

[0141] The bottom plates of all these recesses (agitator recess 1420, channel recess 1430, storage container tank recess 1440) may be planar and coplanar with each other and may be substantially horizontal. The locations where the various recesses (agitator recess 1420, channel recess 1430, storage container groove recess 1440) intersect each other may be provided with rounded corners to improve the smoothness of fluid flow. Rounded internal corners may also be provided at various locations associated with the recesses.

[0142] A drain recess 1450 may also be provided, which may be in fluid communication with the channel recess 1430 and may extend to a lower level than the channel recess 1430. Fluid may be removed from the storage container 1060 by placing a tube into the drain recess from above and pumping out the fluid.

[0143] Now refer to Figure 14-16C, a riser conduit 500 may also be provided that may define a flow path in an upward direction away from the storage vessel 1060. The riser conduit 1500 is shown as having a rectangular cross-section, but it is understood that other cross-sectional shapes are possible. The riser conduit 1500 may have a flat and horizontal riser conduit lower edge 1510. First, the riser conduit 1500 may be positioned such that the riser conduit lower edge 1510 is at a height lower than the height of the liquid level in the storage vessel 1060. More specifically, the riser conduit 1500 may be positioned such that the riser conduit lower edge 1510 is located at a height above the floor of the storage vessel sump recess 1440, but below the upper surface 1410 of the storage vessel base 1400. A storage vessel sump recess 1440 may also be provided that has a boundary that is larger than the riser conduit 1500 and outside the perimeter of the riser conduit 1500. This combination of dimensional relationships can provide an inflow pattern for flow from the storage vessel 1060 to the riser conduit that is distributed substantially around the entire perimeter of the riser conduit 1500. This flow pattern maintains access to a majority of the liquid in the storage vessel 1060 while not creating a "pinch" in the fluid flow with excessively high local fluid velocities or local shear rates.

[0144] Continuing upward in the general direction of riser conduit 1500, there may be a rotor chamber 1600 and a rotor 1700, generally contained within rotor chamber 1600. Further details regarding rotor 1700 are provided elsewhere herein.

[0145] The rotor chamber 1600 may have a vertical passage defined by a lower passage opening 1610 and an upper passage opening 1620. The lower passage opening 1610 and the upper passage opening 1620 may have substantially the same internal cross-sectional area and dimensions. The internal cross-sectional area and dimensions of the lower passage opening 1610 and the upper passage opening 1620 may at least approximately match the internal cross-sectional area and dimensions of the interior of the riser conduit 1500 and the passage through the rotor 1700 (described elsewhere herein).

[0146] The rotor chamber 1600 may rest on or may be mounted on a cover plate of the storage container 1060 .

[0147] Near the top of the rotor chamber 1600, the rotor chamber wall that contacts the liquid may terminate at a flat and horizontal top edge 1630. The top edge 1630 may act as a weir for the liquid to flow over it. Outside the wall may be an overflow moat 1640. The design of the overflow moat 1640 and nearby features may be such that the fluid in the rotor chamber 1640 must flow upward, then flow over the top edge 1630 of the rotor chamber wall, and then flow downward into the moat 1640. The moat 1640 may extend around the entire perimeter of the rotor chamber 1600. During system operation, air pockets may exist above the top edge 1630.

[0148] The rotor chamber 1600 can be supported by a rotor chamber cover 1660, which can be detachable. There may be a gap between the top edge 1630 and the rotor chamber cover 660. The gap can be suitable for flowing through the gap, and therefore the flow can enter the overflow trench 1640 and be collected. It is believed that, although it is not desired to be limited to this explanation, the use of the overflow trench 1640 can help improve the uniformity of the flow through the cell culture area and the tissue scaffold. As another embodiment of this article, there can be an overflow trench 1640, as well as a corresponding storage tank and a nozzle.

[0149] Rotors and cell culture stands

[0150] Within rotor chamber 1600, a rotor 1700 may be provided. Rotor 1700 may be capable of rotating about an axis of rotation relative to rotor chamber 1600. The axis of rotation may be horizontal and may be generally perpendicular to the direction of riser conduit 1500. Rotor 1700 may fit tightly within rotor chamber 600 while still being free to rotate.

[0151] One end of the rotor 1700 may have or may be attached with a shaft to guide the angular position of the rotor 1700. The shaft may in turn be operated by a motor. The motor may be located inside the incubator 1950, or may be located outside the incubator 1950 with the shaft penetrating through the wall of the incubator 1950. The motor may be controlled by a computer or automated system.

[0152] The rotor 1700 may be capable of rotating through various rotational positions. One position of the rotor may correspond to a fully open flow path for flowing through the interior of the rotor and the tissue support. Another position of the rotor 1700 may cause the flow path to be blocked by a substantially solid portion of the rotor 1700.

[0153] Continue to refer Figure 14-16C , showing a rotor 1700 and some associated components. The rotor 1700 can be generally cylindrical except for the absence of material as described herein. The rotor can have two opposing rotor openings 1710, 1712 that define a passage through the rotor 1700, and the rotor can have two other opposing sides 1714, 1716 that are mostly solid. Going around the periphery of the rotor 1700, there can be an open space; a generally solid portion of which the exterior can be cylindrical; an open space; and another generally solid portion of which the exterior can be cylindrical. On the exterior, the generally cylindrical shape can correspond to the interior cylindrical space within the rotor chamber 1600. When the rotor 1700 is in the proper angular position, the dimensions of the mostly solid sides can completely block the riser conduit 1500 (except for the hole 1790). The rotor end can have an inwardly facing groove 1720. The groove 1720 in the rotor 1700 can be appropriately sized to receive and support the edge of a bracket or screen.

[0154] One of the rotor openings 1710 may have a bridge 1740 spanning the rotor opening. Another of the rotor openings 1712 may have a removable clip 1760 spanning the rotor opening. The bridge 1740 and the clip 1760 may be sized so that they only take up a small portion of the total length of the rotor 1700 so as not to substantially interfere with the flow of liquid. The bridge 1740 and the clip 1760 may have internal grooves that correspond to the internal grooves 1720 at the end of the rotor 1700. In combination, the grooves at one end of the rotor 1700, the grooves at the other end of the rotor 1700, the grooves in the bridge 1740, and the grooves in the clip 1760 may all be aligned with each other and may cooperate with each other to support the screen.

[0155] Within the open space of rotor 1700 , a cell growth support, such as mesh 1300 described elsewhere herein, may be provided, and flow may flow over or through the cell growth support (eg, mesh 1300 ).

[0156] In the solid portion of the rotor 1700 having a generally cylindrical exterior, one or more holes 1790 may be provided on each of two opposing sides therethrough. The holes 1790 may be aligned with one another. During the period when the support is in a horizontal orientation, the holes 1790 may allow mass transfer between the liquid inside the support area of ​​the rotor 1700 and the liquid in the storage container. Even when the support (screen) is horizontal, there may not be a large amount of flow of liquid, even if only by diffusion or some form of natural convection, the holes 1790 will still allow some mass transfer.

[0157] The open space through the rotor 1700 may correspond in size and cross-sectional area to the interior size and cross-sectional area of ​​the riser conduit 1500. For example, the dimension from the interior of one substantially solid portion of the rotor 1700 to the dimension of another substantially solid portion of the rotor 1700 may be the same or approximately the same as the interior dimension of the riser conduit 1500 in the same direction. The end-to-end dimension of the empty space of the rotor may be the same or approximately the same as the interior dimension of the riser conduit 1500 in the same direction.

[0158] The open space through the rotor 1700 can be a space intended to be occupied by an array of cell culture supports 1300. The inner face of the rotor 1700 toward the end of the open space may include a groove 1720 or a similar interface to hold multiple cell culture supports. As shown, the ends of the rotor 1700 each have grooves 1720 for 10 individual screens 1300. An alternative design may have grooves for some other number of screens 1300 as needed. The groove 1720 may support or guide or position the end of the screen 1300 at the edge of the screen 1300. The groove 1720 may be appropriately sized for the thickness of each support or screen 1300 and the expected spacing between the supports or screens 1300. At an additional position such as the middle between the two ends of the rotor 1700, a small auxiliary support may be provided, which also includes a groove 1720 to support or guide or position the screen or support 1300 at its edge. For example, a bridge 1740 may be provided that may be connected to the rotor 1700, which may include the groove 1720. A detachable clip 1760 may be provided that may be attached to and detached from the rotor 1700, and may also include the groove 1720.

[0159] The design of the support and screen may be as discussed elsewhere herein in conjunction with another embodiment.

[0160] As shown, the rotor 1700 includes grooves 1720 adapted to hold the stent 1300 so as to flow in a direction parallel to the surface of the stent 1300. Alternatively, a rotor 1700 may be provided that holds the stent 1300 in a position for flow past the stent (perpendicular to the surface of the stent). The device may be designed to accept a variety of different rotors 1700, wherein different rotors 1700 have different designs with respect to the placement or orientation of the stent 1300. With different interchangeable rotors, one rotor may provide flow generally parallel to the surface of the stent, while another different rotor may provide flow past the stent (i.e., generally perpendicular to the stent).

[0161] Considerations related to volume and efficient use of cells and media

[0162] Reference now Fig.17Various considerations may be built into the design and operation of the apparatus to aid in the efficient use of culture medium, which may be an expensive material, and the efficient use of cells, which tend to sink under the influence of gravity. The lower edge 1510 of the riser conduit 1500 may extend below the surface of liquid submerged in the storage vessel 1060, or specifically may extend below the surface of liquid submerged in the storage vessel sump recess 1440 prior to operation and at any time during operation. This may aid in the ability to draw fluid upward. In order to be able to draw liquid upward into the riser conduit 1500, it is preferred to avoid any air traveling below the lower edge 1510 of the riser conduit 1500.

[0163] The available volume of storage vessel 1060 may be defined as the volume of liquid present in storage vessel 1060 consistent with the requirement that the liquid level is below the sprinkler and below the plug in the fill port, and that the liquid level in the riser conduit is the same as the liquid level in storage vessel 1060 outside of riser conduit 1500 when the apparatus is balanced and not in operation. The available volume of storage vessel 1060 may be sufficient such that when storage vessel 1060 contains a suitable amount of liquid that is less than the available geometric volume of storage vessel 1060, storage vessel 1060 is still not empty, or at least storage vessel sump recess 1440 is not empty, even if rotor chamber 1600, moat 1640, and various conduits are full of liquid. More specifically, under these conditions, the liquid level in storage vessel 1060 or storage vessel sump recess 1440 is still at a higher elevation than lower edge 1510 of riser conduit 1500.

[0164] In the absence of a screen in rotor 1700, the volume of storage container 1060 may be sufficient to fill all of these just described spaces with liquid. Alternatively, the volumes of the various components may be calculated and corrected for the amount of space occupied by the screen of the cell culture scaffold, and when the scaffold is present, the volume of the storage container may be sufficient to fill all of these just described spaces. The volume of liquid actually loaded into storage container 1060 may be measured accurately enough so that when the empty spaces such as riser conduit 1500, rotor chamber 1600, trench 1640, and various conduits are filled with liquid, the liquid is sufficient to reach the top of wall 1630, or at least cover the scaffold with liquid, and the liquid level in storage container 1060 may be located in storage container recess 1440 but above lower end 1510 of riser conduit 1500.

[0165] It has been shown that the upper surface 1410 of the storage container base 1400 is flat and horizontal. Alternatively, some slight slope or funnel shape can be provided to help drain the liquid or help the liquid reach the storage container sump recess 1440.

[0166] It can be noted that pump 1140 (e.g., a peristaltic pump) is located downstream of the cell culture chamber, i.e., rotor chamber 1600. This means that during the inoculation of the scaffold, cells can reach the scaffold directly from storage container 1060 without passing through the pump. The passage of cells through the pump may damage the cells undesirably. However, it is expected that during inoculation, most of the cells occupying the cell scaffold area will deposit themselves on the scaffold. It is expected that only a small portion of cells will not be inoculated and will leave rotor chamber 1600 and pass through pump 1140. If those cells that fail to attach to the scaffold are damaged when passing through pump 1140. In any case, the number of these cells should be small, so the possible damage to those unattached cells should not have too much overall significance in terms of the efficiency of using cells.

[0167] The direction of rotation of the stirring rod may be such that it causes liquid in the channel recess 1430 to flow towards the riser conduit 1500. In this way, particularly when the liquid level in the storage vessel 1060 is low, it will provide some useful pumping action in addition to the stirring action.

[0168] Filling structure in the riser

[0169] Reference now Fig.18 In an embodiment of the present invention, a filling structure 1100 may be provided within a riser conduit 1500 .

[0170] The filling structure 1100 can usefully occupy a portion of the volume inside the riser conduit 1500 so that the portion of the volume does not have to be occupied by culture medium liquid. Because culture medium liquid is expensive, and the liquid culture medium in the riser does not contribute to cell seeding or culture, the filling structure 1100 can reduce the amount of such culture medium that cannot be used for useful purposes.

[0171] The filling structure 1100 can have a streamlined shape so as to receive the flow of culture medium entering the bottom of the riser at the riser sump and direct the flow upward and distribute it to the cell culture area. Thus, the upper end of the filling structure 1100 can be directional, pointing vertically upward. The lower end of the filling structure 1100 can be so as to receive flow having a horizontal velocity component from the storage container sump recess 1440 and redirect the flow upward.

[0172] In other embodiments of the present invention, a filling structure similar to the filling structure 1100 may also be used. Even if it is not for saving the amount of liquid culture medium, it may be beneficial to improve the flow pattern, such as improving the uniformity of the flow rate into the mesh array.

[0173] There may be a plurality of fluid containers external to the incubator 1950. Such fluid containers may include waste bottles; recovery bottles; medium storage bottles; PBS (phosphate buffered saline) bottles; and tryp-LE E bottles. Appropriate passages or penetrations may exist to allow these fluids to enter and exit the incubator 950. Appropriate valves may also be provided to control the flow of such fluids. As described elsewhere herein, a motor (e.g., a rotor) capable of rotating the cell culture components may also be provided at this location, or substantially at any desired location.

[0174] Impactor

[0175] In an embodiment of the present invention, an actuator or impactor (not shown) may also be provided, which may be suitable for directing motion within the incubator 1950 or delivering impact to a device within the incubator 1950, such as the outside of the rotor chamber. The vibration caused by the impactor hitting the components near the cell culture screen can break free of the cells and can help harvest the cells after expansion. In the embodiment shown, the impactor can be located at the rear of the incubator 1950. There may be an appropriate pass or penetration for the impactor. This impact can be combined with a chemical treatment (e.g., exposing the cell population to trypsin).

[0176] The apparatus may also include automatic controls suitable for operating the components.

[0177] How to operate

[0178] In embodiments of the present invention, cell seeding and growth on scaffolds can be performed by various protocols.

[0179] During inoculation, the liquid in storage container 1060 may contain cells to be inoculated. During cell expansion, the liquid in storage container 1060 does not necessarily contain cells, but it may contain nutrients that aid in cell growth and proliferation.

[0180] It is expected that during the inoculation phase, the cell / medium mixture will not pass through the pump 1140 substantially. At the beginning of the automatic inoculation process, the cells and the culture medium are in the storage container 1060. The culture medium volume at this time is enough to fill the riser and the cell culture area, but not more than this. When the pump 1140 is started, the cell / medium mixture will be sucked into the cell culture area. It is only necessary to aspirate enough culture medium to cover the cell culture support (screen) with liquid. It is not necessary to fill the trench 1640 with liquid, and it is not necessary to aspirate the liquid far enough to make the liquid enter the tube from the trench 1640 to the pump 1140. This operation can be performed so that when the rotor chamber 1600 is full of the cell / medium mixture, the pump 1140 will stop. For example, using a peristaltic pump, if the rotor of the peristaltic pump stops rotating, there will be no further flow and no backflow. Therefore, the pump 1140 can be simply turned off and the liquid can be allowed to remain at the position (including the liquid level in the culture area) at that time. It is possible that the volume of liquid has been calculated and measured accurately enough so that the storage container 1060 is nearly empty at that time. In fact, the liquid level in the storage container 1060 may have dropped to somewhere in the storage container sump recess 1440. If the liquid level is in the storage container sump recess 1440, below the upper surface 40 of the storage container base 1400 but above the bottom end of the riser conduit 1500, the liquid in the riser conduit 1500 and the cell culture area will remain in a stable position because no bubbles enter the liquid column to allow the liquid to fall. By the reduced gas pressure above the cell culture area (which can be slightly below atmospheric pressure), the cells / medium will be maintained in the riser column 1500 and the cell culture area, even if the pump 1140 is stopped at that time. A pump such as a peristaltic pump or generally a positive displacement pump can maintain this condition. A valve can also be used.

[0181] During the inoculation process, the culture medium containing cells can be lifted into the region of the rotor 1700 and the cell culture support by suction. For the initial inflow of liquid, the position of the screen 1300 can be roughly vertical. Then the inoculation process can be started by rotating the rotor 1700 so that the screen 1300 occupies a horizontal position. This allows the cells suspended in the liquid between the screens 1300 to settle down to the adjacent screen 1300 by gravity. When this happens, the support (screen) 1300 can remain stationary for a period of time. At the desired time, the rotor 1700 can also be rotated again. For example, the rotor 1700 can be rotated 180 degrees so that due to the change in the direction of gravity, cells that may only be placed on the top of a screen 1300 without attachment may float, and settle and attach to the nearby screen 1300. The rotor 1700 can be rotated periodically to produce a uniform cell distribution on the screen or support. This process can last up to one to two days (determined by experiment).

[0182] When inoculation is complete, it is expected that all (or most) cells will be attached to the support (screen) 1300. Subsequent steps may involve more continuous culture medium flow and may be referred to as dynamic culture. Additional culture medium may be added to the storage container 1060. The additional culture medium does not have to contain cells. Pump 1140 may be turned on to allow dynamic culture by culture medium circulation. If desired, flow may flow continuously around the flow system through the support. The flow rate may be adjusted to be low enough to maintain the shear rate at the cell position at a desired small amplitude. At this point, there may be some cells that are not attached to the support and flow through the pump tube. It is desirable to achieve at least 80% inoculation efficiency during inoculation.

[0183] The procedure for operating the motor and positioning the support screen may be as follows:

[0184] • Add the liquid containing the cells to be cultured and expanded to the storage container 1060.

[0185] For initial filling of the culture area with liquid, place the screen vertically.

[0186] After the initial filling of the culture area with liquid, the mesh (rack) 1300 is placed horizontally so that cells suspended between the meshes can settle from the suspension onto the meshes.

[0187] At regular intervals, rotate the screen 180 degrees so that the screen surface facing upwards becomes the screen surface facing downwards and vice versa. The interval may be, for example, one hour.

[0188] Continue doing this long enough so that most of the cells are attached to the mesh 1300. It is expected that this step may take about one to two days.

[0189] After the cells are well seeded, the screen (support) 1300 is rotated to a vertical position or a horizontal position according to the needs of dynamic culture.

[0190] During dynamic culture, the mesh (support) 1300 is kept in a vertical position or a horizontal position as required. During dynamic culture, nutrients are placed in the culture medium and the culture medium is slowly flowed through the mesh (support) 1300. The flow will be upward and approximately parallel to or through (perpendicular to) the flat surface of the mesh 1300 as required.

[0191] After sufficient time has passed, harvest the cells.

[0192] In general, any desired combination of rotor and support positions can be performed at specific stages of the inoculation and cell culture process. During dynamic culture, the screen can be in a vertical or horizontal position (whichever is desired) during culture, and the flow can be parallel to or through (perpendicular to) the screen.

[0193] Harvesting cells

[0194] In an embodiment of the present invention, certain techniques can be implemented for harvesting cells from the support 1300 after cell growth. It is known that tryp-LE E can be used to harvest cells from the support. In an embodiment of the present invention, in order to harvest cells, the support is washed three times with PBS, and then an appropriate amount of tryp-LE E is added for about 30 minutes. The harvesting of cells can be further assisted by the action of an impactor, which impacts the cytoskeleton of cells connected to the screen (or support) on the structure. This impact can detach the cells by the mechanical force of the impact.

[0195] Computational Fluid Dynamics

[0196] Reference now Figures 19A-20 , showing the results of computational fluid dynamics modeling of the flow geometry of an embodiment of the present invention. Fig.19A The local velocity distribution is shown at a flow rate of 30 ml / min (cross-sectional area in the incoming channel is approximately 6200 mm2). Fig.19B The rate profile for a flow rate of 40 ml / min is shown. Fig.19C The rate profile for a flow rate of 60 ml / min is shown. Fig. 20 Pressure distribution and flow streamlines between screens constructed as described herein are shown.

[0197] Further comments

[0198] In general, any combination of the disclosed features, components and methods described herein is possible.The steps of a method may be performed in any order that is physically possible.

[0199] All cited references are incorporated herein by reference.

[0200] While embodiments have been disclosed, the present invention is not intended to be limited thereby. Rather, the scope should be determined only by the following claims.

Claims

1. A bioreactor system, include: a storage container for containing liquid culture medium; a manifold assembly including an upper manifold and a lower manifold, the lower manifold having a lower end extending into the storage container; a scaffold, the scaffold being contained within the manifold assembly and the scaffold retaining a plurality of fiber assemblies suitable for cell growth thereon; and a circulation system for flowing liquid culture medium through the lower manifold and the fiber assembly and the upper manifold, wherein the liquid culture medium flows through in a sequence comprising (a) one of the fiber assemblies, wherein one of the fiber assemblies comprises a plurality of solid fibers connected to other solid fibers at staggered points with the other solid fibers, followed by (b) an open area in which the liquid culture medium flows perpendicular to one of the fiber assemblies through a space not occupied by any solid objects, wherein the sequence repeats itself a plurality of times along the direction of flow of the liquid culture medium, wherein the fiber assembly is retained in the support so that the fiber assembly does not contact any adjacent fiber assembly under static conditions and does not contact any adjacent fiber assembly under conditions of liquid culture medium flow during cell culturing, wherein the upper manifold and the lower manifold and the support have respective internal cross-sectional flow areas that are the same as or within 10% of each other.

2. The bioreactor system of claim 1 , wherein the fiber assembly has a fiber assembly thickness, and the fiber assembly is separated from an adjacent fiber assembly by a spacing distance, a direction of the fiber assembly thickness and a direction of the spacing distance are aligned with each other, and wherein the spacing distance is between 1.5 and 2.5 times the thickness of the fiber assembly.

3. A bioreactor system according to claim 1, wherein at least some of the fiber components include a plurality of fibers forming a first layer and a second layer, the first layer having fibers parallel to each other and oriented in a first direction, the second layer having a plurality of fibers parallel to each other and oriented in a second direction, the second direction being perpendicular to the first direction, and the fibers in the second layer are connected to the fibers in the first layer.

4. A bioreactor system according to claim 1, wherein at least some of the fiber assemblies include a plurality of fibers, and within a single one of the fiber assemblies, the fibers are arranged in at least first, second, third and fourth layers, the fibers in each of the layers are parallel to each other, wherein the fibers in the first layer are parallel to the fibers in the third layer, and the fibers in the second layer are parallel to the fibers in the fourth layer, and wherein when viewed perpendicular to a planar surface of the fiber assembly, the fibers of the third layer are not aligned with the fibers of the first layer, and the fibers of the fourth layer are not aligned with the fibers of the second layer.

5. The bioreactor system of claim 1 , wherein the bending stiffness of the fiber assembly is equal to or greater than a bending stiffness such that if one of the fiber assemblies having a width of 60 mm is simply supported at two points 100 mm apart and loaded with 14.5 grams, the deflection of one of the fiber assemblies at the center span is 3.1 mm.

6. The bioreactor system of claim 1, wherein a cross-sectional flow area of ​​a leakage path including a gap between one of the fiber assemblies and the support is less than 10% of a total flow area of ​​all channels through a single one of the fiber assemblies.

7. The bioreactor system of claim 1 , wherein the lower end of the lower manifold has a first perimeter and the lower end of the lower manifold is at a uniform height around the entire first perimeter, and wherein the upper end of the upper manifold has a second perimeter and the upper end of the upper manifold is at a uniform height around the entire second perimeter of the upper end of the upper manifold.

8. The bioreactor system of claim 1 , wherein the storage container contains contoured fillers therein, at least some of the fillers being lower than the lower end of the lower manifold, the contoured fillers being shaped to guide liquid culture medium to flow smoothly from the storage container into the lower manifold, the contoured fillers being wider at their bottoms and narrower at their tops.

9. A bioreactor system, include: a storage container for containing liquid culture medium; a manifold assembly including an upper manifold and a lower manifold, the lower manifold having a lower end extending into the storage container; a scaffold contained within the manifold assembly, the scaffold holding a plurality of fiber assemblies suitable for cell growth thereon; and a circulation system for flowing liquid culture medium through the lower manifold, the fiber assembly, and the upper manifold, wherein the inner cross-sectional dimension of the lower manifold below the support, the inner cross-sectional dimension of the upper manifold above the support, and the inner cross-sectional dimension of the support are equal to each other, and wherein the side-to-side dimension of the fiber assembly is larger than the corresponding inner dimension of the support, and the side-to-side dimension of the fiber assembly is smaller than the corresponding inner dimension of the manifold assembly where the support is located. 10 . The bioreactor system according to claim 9 , wherein the inner wall of the lower manifold, the inner wall of the upper manifold and the inner wall of the support are parallel and aligned with each other.

11. The bioreactor system of claim 9, wherein the fiber assembly is held in the support such that the fiber assembly does not contact adjacent fiber assemblies under static conditions and does not contact adjacent fiber assemblies under conditions of liquid culture medium flow during cell culture.

12. A bioreactor system according to claim 9, wherein the support comprises two parts that can be engaged with each other by an engagement feature, and wherein the engagement feature does not protrude inwardly beyond the wall of the support, and wherein one of the engagement features has a groove passing therethrough, which is continuous with the groove in the adjacent part of the inner surface of the support.

13. The bioreactor system of claim 9, wherein a portion of the upper manifold is slidably engaged with a portion of the lower manifold, and wherein an upper surface of the support faces a corresponding surface of the upper manifold and a lower surface of the support faces a corresponding surface of the lower manifold.

14. The bioreactor system of claim 9, wherein the storage container is covered by a storage cover and the storage cover has an opening therein, the opening having a raised edge or lip surrounding the opening, and the lower manifold has a flange thereon adapted to contact the lip and the flange extends beyond the lip so that possible condensation on the outer surface of the manifold assembly does not drip into the storage container.

15. The bioreactor system of claim 9, wherein the fiber assembly is constrained within the scaffold but has at least some ability to move relative to the scaffold in at least one direction.

16. The bioreactor system of claim 15, wherein the fiber assembly has at least some ability to move relative to the support in three mutually orthogonal directions.

17. The bioreactor system of claim 9, wherein the support has at least one degree of freedom of movement relative to the lower manifold when the support is disposed within the manifold assembly.

18. A bioreactor system according to claim 9, wherein at least some of the fiber components include a plurality of solid fibers forming a first layer and a second layer, the first layer having fibers parallel to each other and oriented in a first direction, the second layer having a plurality of fibers parallel to each other and oriented in a second direction, the second direction being perpendicular to the first direction, and the fibers in the second layer are connected to the fibers in the first layer.

19. A method for culturing cells, the method comprising: include: A bioreactor is provided, the bioreactor comprising: a storage container for containing a liquid culture medium; a manifold assembly, the manifold assembly comprising an upper manifold and a lower manifold, the lower manifold having a lower end extending into the storage container; a support, the support contained in the manifold assembly, the support holding a plurality of fiber assemblies suitable for growing cells thereon; and a circulation system for flowing the liquid culture medium through the lower manifold and the fiber assemblies and the upper manifold, wherein the liquid culture medium flows through in a sequence, the sequence comprising (a) one of the fiber assemblies, wherein one of the fiber assemblies comprises a plurality of solid fibers connected to other solid fibers at staggered points with other solid fibers, followed by (b) an open area in which the liquid culture medium flows perpendicularly to one of the fiber assemblies through an area not occupied by any solid objects. space, wherein the sequence repeats itself multiple times along the flow direction of the liquid culture medium, wherein the fiber assembly is maintained in the support so that the fiber assembly does not contact any adjacent fiber assembly under static conditions and does not contact any adjacent fiber assembly under conditions of liquid culture medium flow during cell culture, wherein the internal cross-sectional dimensions of the lower manifold below the support, the internal cross-sectional dimensions of the upper manifold above the support and the internal cross-sectional dimensions of the support are equal to each other, and wherein the side-to-side dimensions of the fiber assembly are larger than the corresponding internal dimensions of the support, and the side-to-side dimensions of the fiber assembly are smaller than the corresponding internal dimensions of the manifold assembly in which the support is located; inoculating cells onto the fiber assembly; operating the circulation system under conditions suitable for cell proliferation into a plurality of cultured cells; and harvesting the cultured cells from the bioreactor.

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