This document relates generally to the cell culturing arts and, more particularly, to a bioreactor with enhanced gas transfer and thermal regulation.
Bioreactors are frequently used for culturing cells. Within the bioreactor, a certain level of gas transfer between the gas phase (e.g. air or oxygen) and the liquid phase (the cell culture medium) is required to permit optimal cell growth. A supply of oxygen to the cells is mandatory in such gas transfer.
Gas transfer is especially important for a fixed-bed bioreactor, the high cell density growth of which demands a heightened level of oxygen. The amount of oxygen present in the cell culture medium for enhancing cell growth may be assessed using the concept of gas transfer coefficient, or kLa. This value uses the volumetric mass-transfer coefficient that describes the efficiency with which gas can be delivered to the cell culture medium in a bioreactor for a given set of operating conditions and surfaces.
In the past, proposals have been made for enhancing the gas transfer by providing the bioreactor with one or more bubblers or spargers to form small gas bubbles in the cell culture medium. However, when a sparger is used to support a high cell density bioreactor, the speed of bubble input and the need for an extensive amount of bubbles often results in the negative effects of shear and excessive foaming. This can deleteriously cause undue mechanical and oxidative stress to the cells, which can impact yields. Further, foaming can clog filters and other connectors in the bioreactor. In addition, use of spargers in unstructured high cell density fixed bed bioreactors is challenging as the resulting bubbles can cause large “clouds” or of air pockets as the bubbles have no definable path through the fixed bed. Such bubbles negatively affect homogeneity of the bioreactor conditions and, thus, cell growth.
Likewise, certain fixed bed bioreactors provide for liquid-to-gas contact, such as in a headspace above the fixed bed. While current implementations do serve to provide for gas transfer to the circulating liquid media, the residence time of this liquid in contact with the gas has been limited. Gas transfer would be enhanced by increasing such residence time. It would be further enhanced by also creating turbulence in the liquid while in contact with the gas phase.
It may also be desirable to regulate (typically increase) the internal temperature of a bioreactor. In the past, this has been achieved using an external heater, such as a film applied to an outer surface of the bioreactor. As can be appreciated, this approach is inefficient, given that the heat must transfer through the external wall of the bioreactor, which typically has a high thermal resistance (and even if not, the transferred heat may only directly influence a portion of any contained liquid media proximate to the wall interior). Poor contact between the film and the outer wall can also lead to significant losses. Hence, using the current external heating approach, it can take a significant amount of time to cause even a modest temperature increase, and can be challenging to precisely regulate the temperature of the liquid throughout the bioreactor. Internal heating can be challenging for several reasons including cost and complexity. Also, heat can impair cell growth within the bioreactor if directly applied to bed where cells reside.
Accordingly, a need is identified for a bioreactor, particularly a high cell density fixed bed bioreactor, with enhanced gas transfer, and further with optional thermal regulation capabilities.
According to a first aspect of the disclosure, an apparatus for culturing cells comprises a bioreactor including a fixed cell culture bed. An agitator is provided for pumping liquid through the cell culture bed. A container is provided for the agitator, and a first conduit connected to the container.
In one embodiment, the first conduit comprises an injector conduit for delivering gas bubbles into the container. In another embodiment, the first conduit comprises a drain conduit for draining liquid from the bioreactor. In the former case, the apparatus may further include a second conduit comprising a drain conduit for draining liquid from the bioreactor.
In one embodiment, the bioreactor comprises a central chamber and a cover. The first conduit extends from the cover and through the central chamber to an opening of the container. The apparatus may further include a tubular post associated with the container, the first conduit being connected to the tubular post.
The agitator may comprise a, preferably magnetic, impeller rotatably coupled to the tubular post by a bearing. The agitator may include a plurality of curved vanes. Additionally, or alternatively, the agitator comprises a plurality of vanes, each vane of the plurality of vanes having a higher central portion and a lower peripheral portion so as to create a variable height.
The container may comprise a sloped upper portion adapted to accommodate the variable height agitator. The container includes a fitment adapted to form custom-sized bubbles from a gas delivered thereto by the first conduit. The fitment comprises a perforated portion.
The cell culture bed may be adapted to divide/shear any gas bubbles in the liquid. The cell culture bed may comprise a fixed bed, such as a structured fixed bed. The structured fixed bed may comprise alternating layers of one or more cell immobilization layers, and optionally one or more spacer layers. The one or more cell immobilization layers or the one or more spacer layers form paths to divide or shear gas bubbles in the liquid that travel through the fixed bed.
The cell culture bed may be located in an annular outer chamber of the bioreactor adapted to receive a flow of liquid from a pumping action created by rotation of the agitator in the container, or may be positioned in a container having any other shape (such as a container with a square or rectangular cross-section and arranged to allow liquid to flow through the fixed bed from side to side). The apparatus may further include a plurality of cell culture beds arranged in a stacked configuration within an annular outer chamber of the bioreactor.
In any of the foregoing embodiments, the bioreactor may further include a flow extender, possibly with a thermoregulator, as disclosed herein.
According to a further aspect of the disclosure, an apparatus for culturing cells, comprises a bioreactor including a cell culture bed. An agitator is provided for pumping liquid through the cell culture bed. An injector is provided for injecting a gas into the liquid to form first bubbles having a first size, the agitator converting the first bubbles to second bubbles having a second, smaller size than the first size for delivery to the cell culture bed with the liquid.
The agitator is located in a container in fluid communication with the bioreactor, the container including at least one inlet and at least one outlet. The injector may comprise a tube having an open end located upstream of the at least one inlet to the container. A housing having a central chamber associated with the at least one inlet may be located radially inward of the cell culture bed. An open end of the tube may be positioned within the central chamber. The injector may comprise a tube having an open end located in a fluid flow path between the at least one inlet and the at least one outlet.
The cell culture bed may comprise a fixed bed, such as a structured fixed bed. The structured fixed bed may comprise alternating layers comprising one or more cell immobilization layers and one or more spacer layers. A channel may be formed between the one or more cell immobilization layers and one or more spacer layers to divide/shear any gas bubbles in the liquid into a third bubbles having a third yet smaller size than the second size.
In accordance with a further aspect of the disclosure, an apparatus for culturing cells includes a bioreactor including a cell culture bed, means for pumping liquid through the cell culture bed, and means for injecting a gas into the liquid to form first bubbles having a first size, the pumping means converting the first bubbles to second bubbles having a second, smaller size than the first size for delivery to the bed with the liquid. The means for pumping may comprise an agitator. The means for injecting the gas may comprise a tube connected to a gas supply.
Still a further aspect of the disclosure pertains to a method of culturing cells in a cell culture bed. The method comprises providing a liquid with gas bubbles, agitating the liquid to divide the gas bubbles, and delivering the liquid with the divided gas bubbles to the cell culture bed. The providing step may comprise supplying a gas to the liquid upstream of an agitator for performing the agitating step, or supplying a gas to the liquid to a container including an agitator for performing the agitating step.
In one embodiment, the the cell culture bed comprises alternating layers comprising one or more cell immobilization layers and, optionally, one or more spacer layers forming a channel for the liquid through the bed. One or more of the layers perform the step of further dividing the bubbles as the liquid passes through the cell culture bed.
Yet a further aspect of the disclosure pertains to a method for culturing cells. The method comprises a bioreactor including a cell culture bed. An agitator is provided for pumping liquid through the cell culture bed. An injector is also provided for injecting a gas into the liquid to form bubbles having a first size. The agitator is adapted to divide or shear the gas bubbles into a second size smaller than the first size, and the cell culture bed is adapted to divide or shear the gas bubbles into a third size smaller than the second size.
Still a further aspect of the disclosure relates to an apparatus for culturing cells. The apparatus comprises the steps of providing a liquid with gas bubbles having a first size, agitating the liquid to divide the gas bubbles to a second size smaller than the first size, and delivering the liquid with the divided gas bubbles to the cell culture bed, which divides the bubbles to a third size smaller than the second size.
A further aspect of the disclosure pertains to an apparatus for culturing cells comprising a housing for containing a liquid for delivery to a bed for culturing cells, the housing including a zone having a gas. A flow extender is adapted for extending a residence time and causing turbulence in the liquid in the zone.
In one embodiment, the flow extender comprises a frusto-conical structure having surface structures for causing the turbulence. The flow extender may comprise one or more labyrinthian, maze-like or winding passages. The flow extender may comprise a plurality of steps.
The housing may be arranged with an outer chamber for receiving the bed, the chamber being an upper chamber including a headspace being located above a liquid exit of the bed, and further including a central chamber for returning liquid for entering the bed. The housing may be arranged with an outer chamber for receiving the bed, the chamber being a central chamber for returning liquid for entering the bed. The housing may be arranged with an inner chamber for receiving the bed, the chamber being an outer chamber for returning liquid for entering the bed.
In one embodiment, the flow extender has a peripheral edge greater in height relative to a radially inward edge of the extender. The housing may comprise an annular chamber including the bed and having a radial dimension X, and wherein a radial dimension Y of the extender is greater than 0.5X. The flow extender may be capable of being heated.
Still further, an apparatus for culturing cells is provided. The apparatus comprises a housing for containing a liquid for delivery to a bed for culturing cells, the housing including a headspace having a gas. A flow extender is adapted to extend a residence time in the headspace. The flow extender may comprise a frusto-conical plate and/or one or more maze-like or winding passages. The flow extender may comprise a plurality of steps.
In one embodiment, the housing is arranged with an outer chamber for receiving the bed, the headspace being located above a liquid exit of the bed, and further including a central chamber for returning liquid for entering the bed. The flow extender may have a peripheral edge greater in height relative to a radially inward edge of the extender. The housing may comprise an annular chamber including the bed and having a radial dimension X, and wherein a radial dimension Y of the extender is greater than 0.5X. In any disclosed embodiment, the flow extender, if present, may be heated.
This disclosure also relates to an apparatus for culturing cells including a housing for containing a liquid for delivery to a bed for culturing cells, the housing including a centrifugal pump for pumping liquid through the bed and into a headspace. A flow extender is adapted for increasing a residence time of the liquid in the headspace. An injector is provided for injecting a gas to the liquid to form first bubbles having a first size, the centrifugal pump converting the first bubbles to second bubbles having a second, smaller size than the first size for delivery to the bed with the liquid being pumped.
Still another portion of this disclosure relates to an apparatus for culturing cells comprising a housing for containing a liquid for delivery to a bed for culturing cells. A thermoregulator is located within the housing for regulating a temperature of the liquid without directly heating or cooling the bed.
In one embodiment, the thermoregulator comprises a stepped structure. The apparatus may further include a structure having upstanding walls thermally regulated by the thermoregulator. The thermoregulator may be located in a headspace of the housing. The thermoregulator may include a power source external to the housing and a resistive element internal to the housing. The thermoregulator may comprise a radiator.
Still another part of the disclosure relates to an apparatus for culturing cells. The apparatus comprises a housing for containing a liquid for delivery to a bed for culturing cells, the housing including a headspace having a gas. A flow extender is adapted for extending a residence time and/or creating turbulence in the liquid in the headspace. In one embodiment, the flow extender comprises a surface having a slope of 1-85° relative to a horizontal plane.
Yet another portion of the disclosure relates to an apparatus for culturing cells. The apparatus comprises a housing for containing a liquid for delivery to a bed for culturing cells, the housing including a zone having a gas. A flow extender is adapted for extending a residence time of the liquid in the zone wherein the flow extender comprises a surface having a slope of 1-85° relative to a horizontal plane. In one embodiment, the zone comprises a headspace.
This disclosure also relates to an apparatus for culturing cells. The apparatus comprises a housing for containing a liquid for delivery to a bed for culturing cells, the housing including a zone having a gas. A flow extender is adapted for extending a residence time and/or causing turbulence in the liquid in the zone, the flow extender comprising an open shell.
In one embodiment, the open shell comprises a chamber for receiving a portion of the liquid. A regulator may be provided for regulating flow into the chamber. The open shell may comprise a sloped upper surface. A thermoregulator may be connected to the open shell.
Reference is now made to
Within the interior compartment of the bioreactor housing 112, several compartments or chambers may be provided for transmitting a flow of liquid, gas, or both, throughout the bioreactor 100. As indicated in
As a result of the agitation and pumping action provided by the agitator (impeller 118), liquid may then flow upwardly (as indicated by arrows A in
Liquid exiting the chamber 120 is passed to a “headspace” formed in a chamber 124 between one (upper) side of the bed 122 and the cover 114, where the liquid (media) is exposed to a gas (such as oxygen). In some embodiments, liquid may then flow radially inwardly to a central chamber 126 to return to the lower portion of bed 122. In some embodiments, this central chamber 126 can be columnar in nature and may be formed by an imperforate conduit or tube 128 or rather formed by the central opening of the structured spiral bed.
The chamber 126 returns the liquid to the first chamber 116 (return arrow R) for recirculation through the bioreactor 100, such that a continuous loop results (“bottom to top” in this version). In some embodiments, a sensor, for example a temperature probe or sensor T may also be provided for sensing the temperature of the liquid flowing or residing in the chamber 126. In some embodiments, additional sensors (such as, for example, pH, oxygen, dissolved oxygen, temperature, cell density, etc.) may also be provided at a location before the liquid enters (or re-enters) the chamber 116, including for example at the exit location, or top, of the fixed bed 122.
In some embodiments, the mesh structure included in spacer layers 122b forms tortuous paths to steer the cells into the depth of the cell immobilization layers 122a (see cells L in
As shown in
In some embodiments, other structures can be used which form such tortuous paths. For example,
The orientation of the structured fixed bed 122 may be other than as shown in a bioreactor 100 as shown in
As noted above and more evidently indicated in
As noted previously, it may be desirable to increase the amount of gas transfer to the cell culture media liquid while circulating through the bioreactor 100. According to one aspect of the disclosure, and with reference to
In some embodiments, as shown in
In any of the embodiments, oxygen-containing gas (such as air) injected into the bioreactor 100 via tube 142 forms relatively large size of bubbles in the fluid since the tube simply includes an unobstructed open end. Alternatively, smaller bubbles can be generated with the use of a device such as a sparger is used along with customized nozzles and related oscillator systems. In either case, as a result of the positioning of the open end or nozzle of the tube 142, these larger “macrobubbles” or smaller “microbubbles” (Size 1) are released to a location before which they will encounter the turbulence and shearing action created by the agitator (e.g., rotating impeller 118 serving as agitator) forming the pump for the bioreactor 100. This agitation serves to divide the larger macrobubbles into more numerous bubbles having a smaller size, or “microbubbles” or to divide the smaller microbubbles into yet more numerous bubbles having yet smaller size (Size 2). The formation of more numerous bubbles may further result from the increased residence time of the gas bubbles within the agitator container 140 (if present) as well as the speed and design of the agitator.
As a result of the rotation of impeller 118 to create the flow pattern for the bioreactor 100 shown in
Given their smaller size, the microbubbles are better able to pass into and through channels formed by the spacer layers 122b and the adjacent cell immobilization layers 122a (or other available paths) of the fixed bed 122. This serves to further enhance the oxygenation of the cells being grown in the bed, without a corresponding need to increase the speed of the impeller 118 and the resulting liquid flow rate. Moreover, the release of the gas into or near the agitator container 140 and the resulting flow avoids the creation of deleterious air pockets in the bioreactor 100, which are notoriously difficult to remove without halting the bioreactor operation.
However, in another embodiment of the disclosure, as the (Size 2) microbubbles pass through the structured fixed bed 122, they are further divided into smaller and more numerous microbubbles (Size 3). For example, this may occur as the bubbles enter into the channels formed in the fixed bed 122, such by the spacer layers 122b and/or the adjacent cell immobilization layers 122a and pass through all available tortuous (or labyrinthian) paths provided. As they travel, the larger bubbles tend to break up or divide into still yet smaller microbubbles before exiting the fixed bed 122. Consequently, the gas transfer during residence time in the fixed bed 122 is increased and the bubbles emanating from the bed once having passed therethrough are actually still even smaller in size (and thus more numerous) that at a point of entry, as a result of shearing forces caused by the bubbles engaging the mesh of the spacer layer 122b in the illustrated embodiment.
As indicated in
A further embodiment of a bioreactor 100 is shown in
As perhaps best understood with reference to the cross-sectional, enlarged view of
Thus, gas delivered via tube 142 is introduced into the container 140 as bubbles of a first size created by holes 115. These gas bubbles when introduced into liquid within the container 140 flow directly into the path of the impeller 118 when rotating. As referenced hereinabove, contact with the rotating impeller 118 causes shearing of the bubbles, which tends to reduce their size. As a result of the pumping action created, the associated liquid with the smaller-sized bubbles as a result of the shearing action created by engagement with the rotating impeller 118 is then ejected radially outwardly from the container 140 via openings 141 and flows with the associated liquid into (vertically through) the one or more fixed beds of the bioreactor 100.
As noted above, in the case of a structured fixed bed comprising layers spaced in a radially outward direction, the microbubbles are better able to pass into and through channels formed by the spacer layers (not shown in
Turning now to
Referring to
As can be appreciated from
The lower wall 154 may also connect to an upstanding wall 156 adapted to seal with the central column of the bioreactor, such as by snap fit engagement. Thus, liquid flow is urged into a central opening 159 in the wall 154 to enter the container 140. An anti-vortex structure, such as for example in the form of upstanding generally square panels 161 (e.g., four spaced 90 degrees apart about the axis X), may also be provided adjacent to the opening 159, and may form part of the lower wall 154.
The table in
Additionally (or alternatively), increasing the gas transfer coefficient, or kLa, value may be achieved by increasing or extending the distance, and thus the time during which, the liquid media travels while in contact with gas phase prior to returning to the fixed bed. In one possible embodiment, this extended flow pattern is achieved by providing a flow extender (which for purposes of this disclosure means a structure with an increased surface area over which the liquid must flow in order to increase the residence time in contact with gas, and may be formed of any suitable material, such as polymer, metal, or the like). It may also be desirable, as discussed further below, to combine the flow extender with a flow disruptor to cause turbulence, which minimizes the diffusion layer normally formed at the gas/liquid border. One skilled in the art will appreciate that this introduction of a flow extender and/or flow disrupter can be applied to any fixed bed bioreactor design including an unstructured packed bed bioreactor.
The flow extender 150 in this embodiment has a radial dimension Y. This radial dimension Y may be at least 50% of the radial dimension X of the annular chamber 120, but instead may be anywhere between 1-99% of X. In this manner, the liquid flow is diverted from a more direct path to entering the return (e.g., column forming chamber 126) such that the residence or contact time between the liquid and the headspace gas in the chamber 124 is increased. While the outer edge 150a is shown as being elevated relative to the inner edge 150b, it can be appreciated that the arrangement may be reversed, such as in a situation where the direction of liquid flow is radially outward (but the concept of creating a longer flow path remains the same), as shown in
While the flow extender 150 shown in
As further shown in
In any of the foregoing embodiments, the extender 150 may also be provided with appendages or surface texture to create turbulence in the liquid as it traverses across the corresponding surface. Such turbulence caused thereby increases the opportunity for gas transfer to the liquid media. For example, embosses (e.g., circumferential grooves, radial grooves, or a combination thereof) or bosses (e.g., circumferential projections, radial projections, or a combination thereof) may be provided in the surface for causing deviations in the flow of liquid along the plate.
Likewise, the extender may also include with forks, posts, pins, barriers or like structures to create turbulence in the flow. For example, as shown in
One skilled in the art will appreciate that the gates 170 can be formed of vertical posts as shown but could also be formed by angled or horizontal posts or a combination of any gate- or fence-like structure. The gate(s) 170 thus serve to create further disruptions in the liquid flow, thus increasing the turbulence and improving the gas transfer. As perhaps best shown in
While the above embodiments illustrate the flow extender in association with the zone or chamber 124 including a headspace in the bioreactor 100, it can be appreciated that the extender concept may be applied to other locations in the bioreactor. For instance, as shown in
In a case where the bed 122 is in the outer (annular) chamber 120, the extender 150 also may be located within the central chamber 126, as shown in
Turning back to
Turning to
As shown, this arrangement may benefit from the implementation of various optional features described herein. For example, as shown, a gas may be introduced into the bioreactor 100 by tube 142, such as directly to the container 140 for impeller 118 or to one or more chambers 116, 120, 126 by re-positioning the outlet of the tube 142 and/or providing multiple tubes. In any case, the action of the impeller 118 serves to create shear and divide any gas bubbles into microbubbles, which then enter the bed 112 as a result of the flow pattern created. Given their smaller size, the microbubbles are better able to pass into and through channels formed by the spacer layers 122b and the adjacent cell immobilization layers 122a (or other available paths) of the fixed bed 122. This serves to further enhance the oxygenation of the cells being grown in the bed, without a corresponding need to increase the speed of the impeller 118 and the resulting liquid flow rate.
Indeed, as the microbubbles pass through channels formed by the spacer layers 122b and the adjacent cell immobilization layers 122a (or other available paths in case spacer layer is omitted), they tend to break up into yet smaller microbubbles as they travel therethrough. Hence, the gas transfer during residence time in the fixed bed is increased and the bubbles emanating from the bed 122 once having passed therethrough are actually still even smaller in size that at a point of entry, as a result of shearing forces caused by the bubbles engaging the mesh of the spacer layer 122b in the illustrated embodiment. Moreover, the release of the gas into or near the agitator container 140 and the resulting flow avoids the creation of deleterious air pockets in the bioreactor 100, which are notoriously difficult to remove without halting the bioreactor operation.
The embodiments of bioreactors 100 shown in
As can be appreciated, the flow extender 150 may be integrally formed as part of the bioreactor 100, or may be provided as an optional insert that may be added or removed as necessary or desired. The flow extender 150 is also readily scalable for use in a wide variety and sizing of bioreactors, including other forms not shown in the exemplary drawing figures.
As indicated in
The table of
A further aspect of the disclosure pertains to thermal regulation of the bioreactor and, in particular, thermal regulation of the liquid encountering the cells, which may be achieved using a thermoregulator for regulating the temperature of the liquid within the interior of the bioreactor itself. For example, as shown in
The heater 200 itself may be located partially external to the bioreactor. The heater 200 may be, for example, a resistive heater wherein electrical current is supplied to a resistive element 202 associated with the extender 150, such as along a base thereof including one or more of the steps 158, one or more of the upstanding walls 156, or both. Alternatively, the heater 200 may comprise a radiator, which may comprise a heating element for warming a liquid external to the bioreactor, which is then delivered to the extender 150 (such as via a recirculation loop 204, as shown in
Further examples are illustrated in
In any case, when the liquid contacts the extender 150 and contacts the thermally regulated surfaces (floor and/or walls, which may naturally be heated if in thermal communication with the floor of extender), the temperature of the liquid is regulated (which temperature may be monitored via feedback loop using one or more sensors to ensure adequate control). Of course, the extender 150 functions to increase the residence time of the liquid and/or create turbulent flow, as described previously, and thus helps to ensure that the liquid temperature regulation is reliably achieved as a result of the contact, even if the velocity of the liquid is relatively high (which also helps to prevent overregulating). The extender 150 also has a large surface area, and thus helps to maximize the amount of contact with the liquid and the resulting heat transfer that may be achieved, which may reduce the amount of energy input (especially compared to a film applied to an external wall of a bioreactor). Using the proposed approach, thermal regulation internal to the bioreactor may be achieved in a manner that does not rely on thermal transfer through external walls, since direct contact between the heating element and liquid is achieved. This ensures that the temperature of the liquid may be regulated more efficiently and avoids damage to cells fixed in the bed.
Reference is also made to
As can be appreciated, this increases the total surface area for contacting the flowing liquid, which may enhance the thermoregulation in the case where the surfaces are heated or cooled (which may be done in the ways previously described). The liquid flows may then recombine along the inner periphery, such as for being returned along the central column (not shown) One or both of the surfaces 153, 155 may also be provided with turbulence generating structures. It can also be appreciated that valves may be provided to regulate the amount of liquid entering the chamber 150c, which would thus form a regulator that could be used to control the thermal regulation of the liquid by controlling the amount in contact with the thermally regulated lower surface 150b. It can be appreciated that the liquid flow may also go in the opposite direction (e.g., upward from the central column, radially outwardly, and returning via the outer chamber).
This disclosure may be considered to relate to the following items:
1. An apparatus for culturing cells, comprising:
2. The apparatus of item 1, wherein the first conduit comprises an injector conduit for delivering gas bubbles into the container.
3. The apparatus of item 1, wherein the first conduit comprises a drain conduit for draining liquid from the bioreactor.
4. The apparatus of item 2, further including a second conduit comprising a drain conduit for draining liquid from the bioreactor.
5. The apparatus of any of items 1-4, wherein the bioreactor comprises a central chamber and a cover, the first conduit extending from the cover and through the central chamber to an opening of the container.
6. The apparatus of any of items 1-5, further including a tubular post associated with the container, the first conduit being connected to the tubular post.
7. The apparatus of any of items 1-6, wherein the agitator comprises a, preferably magnetic, impeller rotatably coupled to the tubular post by a bearing.
8. The apparatus of any of items 1-7, wherein the agitator comprises a plurality of curved vanes.
9. The apparatus of any of items 1-8, wherein the agitator comprises a plurality of vanes, each vane of the plurality of vanes having a higher central portion and a lower peripheral portion so as to create a variable height.
10. The apparatus of item 9, wherein the container comprises a sloped upper portion adapted to accommodate the variable height agitator.
11. The apparatus of any of items 1-10, wherein the container includes a fitment adapted to form custom-sized bubbles from a gas delivered thereto by the first conduit.
12. The apparatus of item 11, wherein the fitment comprises a perforated portion.
13. The apparatus of any of items 1-12, wherein the cell culture bed is adapted to divide/shear any gas bubbles in the liquid.
14. The apparatus of any of items 1-13, wherein the cell culture bed comprises a fixed bed.
15. The apparatus of any of items 1-14, wherein the fixed bed comprises a structured fixed bed.
16. The apparatus of any of items 1-16, wherein the structured fixed bed comprises alternating layers comprising one or more cell immobilization layers, and optionally one or more spacer layers.
17. The apparatus of item 17, wherein the one or more cell immobilization layers or the one or more spacer layers form paths to divide or shear gas bubbles in the liquid that travel through the fixed bed.
18. The apparatus of any of items 1-17, wherein the cell culture bed is located in an annular outer chamber of the bioreactor adapted to receive a flow of liquid from a pumping action created by rotation of the agitator in the container.
19. The apparatus of any of items 1-18, further including a plurality of cell culture beds arranged in a stacked configuration within an annular outer chamber of the bioreactor.
20. The apparatus of any of items 1-19, wherein the bioreactor further includes a flow extender, possibly with a thermoregulator.
21. An apparatus for culturing cells, comprising:
22. The apparatus of item 21, wherein the agitator is located in a container in fluid communication with the bioreactor, the container including at least one inlet and at least one outlet.
23. The apparatus of item 21 or item 22, wherein the injector comprises a tube having an open end located upstream of the at least one inlet to the container.
24. The apparatus of any of items 21-23, further including a housing having a central chamber associated with the at least one inlet and located radially inward of the cell culture bed, wherein the open end of the tube is positioned within the central chamber.
25. The apparatus of any of items 21-24, wherein the injector comprises a tube having an open end located in a fluid flow path between the at least one inlet and the at least one outlet.
26. The apparatus of any of items 21-25, wherein the cell culture bed comprises a fixed bed.
27. The apparatus of any of items 21-26, wherein the fixed bed comprises a structured fixed bed.
28. The apparatus of any of items 21-27, wherein the structured fixed bed comprises alternating layers comprising one or more cell immobilization layers and one or more spacer layers.
29. The apparatus of item 28, wherein a channel is formed between the one or more cell immobilization layers and one or more spacer layers to divide/shear any gas bubbles in the liquid into a third bubbles having a third yet smaller size than the second size.
30. An apparatus for culturing cells, comprising:
31. The apparatus of item 30, wherein the means for pumping comprises an agitator.
32. The apparatus of item 30 or item 31, wherein the means for injecting the gas comprises a tube connected to a gas supply.
33. A method of culturing cells in a cell culture bed, comprising:
34. The method of item 33, wherein the providing step comprises supplying a gas to the liquid upstream of an agitator for performing the agitating step.
35. The method of item 33 or item 34, wherein the providing step comprises supplying a gas to the liquid to a container including an agitator for performing the agitating step.
36. The method of any of items 33-35, wherein the cell culture bed comprises alternating layers comprising one or more cell immobilization layers and one or more spacer layers forming a channel for the liquid through the bed, the layers performing the step of further dividing the bubbles as the liquid passes through the cell culture bed.
37. A method for culturing cells, comprising:
38. An apparatus for culturing cells, comprising:
39. An apparatus for culturing cells, comprising:
40. The apparatus of item 39, wherein the flow extender comprises a frusto-conical structure having surface structures for causing the turbulence.
41. The apparatus of item 39 or item 40, wherein the flow extender comprises one or more labyrinthian, maze-like or winding passages.
42. The apparatus of any of items 39-41, wherein the flow extender comprises a plurality of steps.
43. The apparatus of any of items 39-42, wherein the housing is arranged with an outer chamber for receiving the bed, the chamber being an upper chamber including a headspace being located above a liquid exit of the bed, and further including a central chamber for returning liquid for entering the bed.
44. The apparatus of any of items 39-43, wherein the housing is arranged with an outer chamber for receiving the bed, the chamber being a central chamber for returning liquid for entering the bed.
45. The apparatus of any of items 39-44, wherein the housing is arranged with an inner chamber for receiving the bed, the chamber being an outer chamber for returning liquid for entering the bed.
46. The apparatus of any of items 39-45, wherein the flow extender has a peripheral edge greater in height relative to a radially inward edge of the extender.
47. The apparatus of any of items 39-46, wherein the housing comprises an annular chamber including the bed and having a radial dimension X, and wherein a radial dimension Y of the extender is greater than 0.5X.
48. The apparatus of any of items 39-47, wherein the flow extender is capable of being heated.
49. An apparatus for culturing cells, comprising:
50. The apparatus of item 49, wherein the flow extender comprises a frusto-conical plate.
51. The apparatus of item 49 or item 50, wherein the flow extender comprises one or more maze-like or winding passages.
52. The apparatus of any of items 49-51, wherein the flow extender comprises a plurality of steps.
53. The apparatus of any of items 49-52, wherein the housing is arranged with an outer chamber for receiving the bed, the headspace being located above a liquid exit of the bed, and further including a central chamber for returning liquid for entering the bed.
54. The apparatus of any of items 49-53, wherein the flow extender has a peripheral edge greater in height relative to a radially inward edge of the extender.
55. The apparatus of any of items 49-54, wherein the housing comprises an annular chamber including the bed and having a radial dimension X, and wherein a radial dimension Y of the extender is greater than 0.5X.
56. The apparatus of any of items 49-55, wherein the flow extender is heated.
57. An apparatus for culturing cells, comprising:
58. The apparatus of item 57, wherein the flow extender is heated.
59. An apparatus for culturing cells, comprising:
60. The apparatus of item 59, wherein the thermoregulator comprises a stepped structure.
61. The apparatus of item 59 or item 60, further including a structure having upstanding walls thermally regulated by the thermoregulator.
62. The apparatus of any of items 59-61, wherein the thermoregulator is located in a headspace of the housing.
63. The apparatus of any of items 59-62, wherein the thermoregulator comprises a power source external to the housing and a resistive element internal to the housing.
64. The apparatus of any of items 59-63, wherein the thermoregulator comprises a radiator.
65. An apparatus for culturing cells, comprising:
66. The apparatus of item 65, wherein the flow extender comprises a surface having a slope of 1-85° relative to a horizontal plane.
67. An apparatus for culturing cells, comprising:
68. The apparatus of item 67, wherein the zone comprises a headspace.
69. An apparatus for culturing cells, comprising:
70. The apparatus of item 69, wherein the open shell comprises a chamber for receiving a portion of the liquid.
71. The apparatus of item 69 or item 70, further including a regulator for regulating flow into the chamber.
72. The apparatus of any of items 69-71, wherein the open shell comprises a sloped upper surface.
73. The apparatus of any of items 69-72, further including a thermoregulator connected to the open shell.
As used herein, the following terms have the following meanings:
“A”, “an”, and “the” as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a compartment” refers to one or more than one compartment.
“About,” “substantially,” “generally” or “approximately,” as used herein referring to a measurable value, such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/−20% or less, preferably +/−10% or less, more preferably +/−5% or less, even more preferably +/−1% or less, and still more preferably +/−0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier “about” refers is itself also specifically disclosed.
“Comprise”, “comprising”, and “comprises” and “comprised of” as used herein are synonymous with “include”, “including”, “includes” or “contain”, “containing”, “contains” and are inclusive or open-ended terms that specifies the presence of what follows, e.g., “component includes” does not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.
While preferred embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. For example, while the bioreactor is shown in a vertical orientation, it could be used in any orientation. The bioreactor may also be formed of rigid, flexible, or semi-flexible materials, and may be made for single or multiple uses. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the protection under the applicable law and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application claims priority to U.S. Provisional Patent Application Ser. No. 62/942,345, filed Dec. 2, 2019, and U.S. Provisional Patent Application Ser. No. 63/004,706, filed Apr. 3, 2020, the disclosures of which are incorporated herein by reference. This application further incorporates by reference U.S. Provisional Patent Application Ser. Nos. 62/758,152, 62/733,375, and 62/608,261; U.S. Patent Application Publication No. 2018/0282678; International Patent Application PCT/EP2018/076354; U.S. Provisional Patent Application 62/711,070; and U.S. Provisional Patent Application 62/725,545.
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/EP2020/084317 | 12/2/2020 | WO |
Number | Date | Country | |
---|---|---|---|
63004706 | Apr 2020 | US | |
62942345 | Dec 2019 | US |