Mixing assemblies comprising inflatable chambers, associated systems, and methods

Abstract

Mixing assemblies comprising inflatable chambers and associated systems and methods are generally provided. The mixing assemblies described herein may have one or more desirable properties, such as being capable of being used with a mixing chamber without being exposed to the contents therein, being capable of causing desirable mixing to occur, and / or being capable of causing a variety of types of mixing to occur.

Description

[0001] MIXING ASSEMBLIES COMPRISING INFLATABLE CHAMBERS, ASSOCIATED SYSTEMS, AND METHODS

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 702,100, filed October 1, 2024, and entitled “Mixing Assemblies Comprising Inflatable Chambers, Associated Systems, and Methods,” which is incorporated herein by reference in its entirety for all purposes.

[0004] FIELD

[0005] Mixing assemblies comprising inflatable chambers and associated systems and methods are generally provided.

[0006] BACKGROUND

[0007] Some laboratory processes involve mixing two or more components together inside a mixing chamber. However, certain existing processes for mixing have one or more drawbacks, such as requiring contact between a mixing element and the components being mixed and / or resulting in efficient mixing.

[0008] Accordingly, improvements in mixing technology are needed.

[0009] SUMMARY

[0010] The present disclosure generally describes mixing assemblies, systems, and associated methods. The subject matter described herein involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more mixing assemblies, systems, and / or articles.

[0011] Paragraph 1: In some embodiments, a system is provided. The system comprises a mixing chamber containing an internal volume, a mixing assembly comprising an inflatable chamber containing an internal volume, and a flexible component positioned between the internal volume of the mixing chamber and the internal volume of the inflatable chamber. The internal volume of the inflatable chamber is positioned external to the internal volume of the mixing chamber. The flexible component is positioned such that inflation of the inflatable chamber causes local deformation of the flexible component towards the mixing chamber, thereby locally reducing the internal volume of the mixing chamber. The flexible component is positioned such that deflation of the inflatable chamber causes local

[0012] 1

[0013] #14426357vl deformation of the flexible component away from the mixing chamber, thereby locally increasing the internal volume of the mixing chamber.

[0014] Paragraph 2: In some embodiments, a method is provided. The method comprises, in the system of Paragraph 1, inflating the inflatable chamber, thereby locally deforming the flexible component towards the mixing chamber and locally reducing the internal volume of the mixing chamber.

[0015] Paragraph 3: In some embodiments, a method as in any preceding Paragraph further comprises deflating the inflatable chamber.

[0016] Paragraph 4: In some embodiments, a method comprises, in the system of Paragraph 1, deflating the inflatable chamber, thereby locally deforming the flexible component away from the mixing chamber and locally increasing the internal volume of the mixing chamber.

[0017] Paragraph 5: In some embodiments, a method as in any preceding Paragraph further comprises inflating a second inflatable chamber, thereby locally deforming a second flexible component towards the mixing chamber and locally reducing the internal volume of the mixing chamber.

[0018] Paragraph 6: In some embodiments, a method as in any preceding Paragraph further comprises deflating a second inflatable chamber, thereby locally deforming a second flexible component away from the mixing chamber and locally increasing the internal volume of the mixing chamber.

[0019] Paragraph 7: In some embodiments, in a system or method as in any preceding Paragraph, the second flexible component is the same component as the flexible component.

[0020] Paragraph 8: In some embodiments, in a system or method as in any preceding Paragraph, the inflatable chamber is inflated before the second inflatable chamber is inflated.

[0021] Paragraph 9: In some embodiments, in a system or method as in any preceding Paragraph, the inflatable chamber is deflated before the second inflatable chamber is deflated.

[0022] Paragraph 10: In some embodiments, in a system or method as in any preceding Paragraph, the inflatable chamber is inflated while the second inflatable chamber is deflated, thereby causing the internal volume of the mixing chamber, as a whole, to be substantially unchanged.

[0023] Paragraph 11: In some embodiments, in a system or method as in any preceding Paragraph, the inflatable chamber and the second inflatable chamber are capable of being inflated and deflated independently from each other.

[0024] 2

[0025] #14426357vl Paragraph 12: In some embodiments, a method as in any preceding Paragraph further comprises inflating and deflating the inflatable chamber and / or the second inflatable chamber in a pre-determined pattern.

[0026] Paragraph 13: In some embodiments, in a system or method as in any preceding Paragraph, inflating and / or deflating the inflatable chamber and / or the second inflatable chamber causes mixing of a liquid present in the internal volume of the mixing chamber.

[0027] Paragraph 14: In some embodiments, in a system or method as in any preceding Paragraph, the mixing comprises wave-motion mixing.

[0028] Paragraph 15: In some embodiments, in a system or method as in any preceding Paragraph, the mixing comprises rocking-motion mixing.

[0029] Paragraph 16: In some embodiments, in a system or method as in any preceding Paragraph, inflating the inflatable chamber comprises filling the inflatable chamber with a fluid and / or inflating the second inflatable chamber comprises filling the second inflatable chamber with a fluid.

[0030] Paragraph 17: In some embodiments, in a system or method as in any preceding Paragraph, deflating the inflatable chamber comprises removing a fluid from the inflatable chamber and / or deflating the second inflatable chamber comprises removing a fluid from the second inflatable chamber.

[0031] Paragraph 18: In some embodiments, in a system or method as in any preceding Paragraph, the fluid is water.

[0032] Paragraph 19: In some embodiments, in a system or method as in any preceding Paragraph, the fluid is a gas.

[0033] Paragraph 20: In some embodiments, in a system or method as in any preceding Paragraph, the fluid is supplied pneumatically.

[0034] Paragraph 21: In some embodiments, in a system or method as in any preceding Paragraph, the inflatable chamber comprises one or more welds.

[0035] Paragraph 22: In some embodiments, in a system or method as in any preceding Paragraph, the flexible component is positioned directly between the internal volume of the mixing chamber and the internal volume of the inflatable chamber.

[0036] Paragraph 23: In some embodiments, in a system or method as in any preceding Paragraph, the flexible component forms a wall of the mixing chamber.

[0037] Paragraph 24: In some embodiments, in a system or method as in any preceding Paragraph, the flexible component forms a wall of the inflatable chamber.

[0038] 3

[0039] #14426357vl Paragraph 25: In some embodiments, in a system or method as in any preceding Paragraph, the mixing chamber comprises a flexible wall, and wherein the flexible wall of the mixing chamber is mechanically coupled to the flexible component.

[0040] Paragraph 26: In some embodiments, in a system or method as in any preceding Paragraph, the inflatable chamber comprises a flexible wall, and wherein the flexible wall of the inflatable chamber is mechanically coupled to the flexible component.

[0041] Paragraph 27: In some embodiments, in a system or method as in any preceding Paragraph, the flexible component comprises an elastomer.

[0042] Paragraph 28: In some embodiments, in a system or method as in any preceding Paragraph, the elastomer is silicone.

[0043] Paragraph 29: In some embodiments, in a system or method as in any preceding Paragraph, the flexible component comprises a membrane.

[0044] Paragraph 30: In some embodiments, in a system or method as in any preceding Paragraph, the membrane is permeable to a gas.

[0045] Paragraph 31: In some embodiments, a method as in any preceding Paragraph further comprises supplying a gas from an inflatable chamber, through the membrane, and to a liquid present in the internal volume of the mixing chamber.

[0046] Paragraph 32: In some embodiments, in a system or method as in any preceding Paragraph, the mixing chamber is a bag.

[0047] Paragraph 33: In some embodiments, in a system or method as in any preceding Paragraph, the mixing chamber is a flask.

[0048] Paragraph 34: In some embodiments, in a system or method as in any preceding Paragraph, the mixing chamber is a bioreactor.

[0049] Paragraph 35: In some embodiments, in a system or method as in any preceding Paragraph, the mixing chamber has an internal volume having a size of greater than or equal to 10 microliters and less than or equal to 500 L.

[0050] Paragraph 36: In some embodiments, in a system or method as in any preceding Paragraph, a liquid is present in the internal volume of the mixing chamber.

[0051] Paragraph 37: In some embodiments, in a system or method as in any preceding Paragraph, cells are suspended in the liquid.

[0052] Paragraph 38: In some embodiments, a method as in any preceding Paragraph further comprises employing the system to mix chemicals, buffers, and / or cell-culture media.

[0053] Paragraph 39: In some embodiments, a method as in any preceding Paragraph further comprises employing the system during RNA in-vitro synthesis.

[0054] 4

[0055] #14426357vl Paragraph 40: In some embodiments, a method as in any preceding Paragraph further comprises employing the system to mix polyplexes for transfection and / or gene therapy.

[0056] Paragraph 41: In some embodiments, a method as in any preceding Paragraph further comprises employing the system to homogenize post-thaw cryofrozen samples.

[0057] Paragraph 42: In some embodiments, a method as in any preceding Paragraph further comprises employing the system to deliver cell therapy products.

[0058] Paragraph 43: In some embodiments, a system as in any preceding Paragraph is configured to perform and / or capable of performing the method of any preceding Paragraph.

[0059] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

[0060] BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures:

[0062] FIGs. 1-3 show three non-limiting examples of systems comprising a mixing chamber, a mixing assembly, and a flexible component, in accordance with some embodiments;

[0063] FIG. 4 shows one non-limiting example of a system in which a flexible component is positioned directly between the internal volume of a mixing chamber and the internal volume of an inflatable chamber forming part of a mixing assembly, in accordance with some embodiments;

[0064] 5

[0065] #14426357vl FIG. 5 shows one non-limiting example of a system that comprises two flexible components positioned between a mixing chamber (and its internal volume) and a mixing assembly, in accordance with some embodiments;

[0066] FIG. 6 shows a system component that comprises an internal volume contained by walls, in accordance with some embodiments;

[0067] FIG. 7 shows first and second system components for which the internal volume of the first system component is not external to the internal volume of the second system component, in accordance with some embodiments;

[0068] FIG. 8 shows first and second system components for which the internal volume of the first system component is external to the internal volume of the second system component, in accordance with some embodiments;

[0069] FIG. 9 shows the inflation and deflation of a mixing chamber, in accordance with some embodiments;

[0070] FIG. 10 shows one non-limiting example of a method, in accordance with some embodiments;

[0071] FIG. 11 shows one non-limiting example of two inflatable chambers, each comprising a plurality of welds, in accordance with some embodiments;

[0072] FIG. 12 shows an exemplary pair of inflatable chambers, in accordance with some embodiments;

[0073] FIGs. 13-14 show examples of pluralities of inflatable chambers, in accordance with some embodiments;

[0074] FIG. 15 shows one non-limiting example of a mixing chamber taking the form of a bag, in accordance with some embodiments;

[0075] FIGs. 16-21 show various examples of systems comprising mixing chambers taking the form of cell-culture bags that are disposed on pluralities of inflatable chambers, in accordance with some embodiments;

[0076] FIG. 22 shows one example of a chip and associated hardware, in accordance with some embodiments;

[0077] FIG. 23 shows an exemplary diagram of electronic and pneumatic systems that may be employed in the systems described herein, in accordance with some embodiments;

[0078] FIG. 24 shows various mixing programs, in accordance with some embodiments;

[0079] FIGs. 25-26 show the anticipated inflation level of inflatable chambers as a function of time for various mixing programs, in accordance with some embodiments;

[0080] 6

[0081] #14426357vl FIGs. 27-29 show experimental data for mixing as a function of time, in accordance with some embodiments;

[0082] FIG. 30 shows experimental data for cell concentration as a function of time, in accordance with some embodiments; and

[0083] FIG. 31 shows experimental data for cell viability as a function of time, in accordance with some embodiments.

[0084] DETAILED DESCRIPTION

[0085] Mixing assemblies comprising inflatable chambers and associated systems and methods are generally provided. The mixing assemblies described herein may have one or more desirable properties, such as being capable of being used with a mixing chamber without being exposed to the contents therein, being capable of causing desirable mixing to occur, and / or being capable of causing a variety of types of mixing to occur.

[0086] In some embodiments, a mixing assembly causes mixing (and / or is configured to and / or capable of causing mixing) in a mixing chamber via a component that is positioned external to the mixing chamber. This property may desirably allow for the mixing assembly to cause mixing without being exposed to the contents of the mixing chamber. This may beneficial when the contents of the mixing chamber are incompatible with the mixing assembly, the mixing assembly is incompatible with one or more contents of the mixing chamber, and / or the contents of the mixing chamber could contaminate the mixing assembly in a way that would be laborious, challenging, and / or impossible to remedy.

[0087] In some embodiments, a mixing chamber causes mixing by affecting a volume and / or a geometry of an internal volume of a mixing chamber. This may be accomplished by locally deforming a flexible component that is positioned proximate to the internal volume of the mixing chamber. The flexible component may be deformed to increase or reduce the total internal volume of the mixing chamber, and / or it may be deformed in a manner such that the total internal volume of the mixing chamber stays the same, but the shape of the internal volume of the mixing chamber is affected. For instance, the flexible component may be deformed to locally increase the internal volume of the mixing chamber in one direction while simultaneously locally reducing the internal volume of the mixing chamber by the same amount in another direction. It is also possible for a system to comprise two or more flexible components that can be deformed, and, in such instances, such flexible components may be deformed such that their total deformation results in the total internal volume of the mixing chamber remaining the same (even if the total deformation of one or more of the

[0088] 7

[0089] #14426357vl flexible components alone results in a net increase or decrease in the total internal volume of the mixing chamber).

[0090] Changing the internal volume of the mixing chamber and / or the shape of the internal volume of the mixing chamber may cause one or more components present therein to undergo flow and / or mix. For instance, locally reducing the internal volume of the mixing chamber may cause a component initially present therein to be removed from and / or flow away from the portion of the internal volume being reduced. As another example, locally increasing the internal volume of the mixing chamber may cause a component initially present herein to be introduced to and / or to flow towards the portion of the internal volume being increased. Such flow and / or movement may cause mixing.

[0091] In some embodiments, a system described herein comprises a mixing chamber, a mixing assembly, and a flexible component. As described in further detail below, such components may be employed together to mix a component contained in the mixing chamber.

[0092] In some embodiments, a mixing chamber contains an internal volume. Such an internal volume may contain, be capable of containing, and / or be configured to contain a component to be mixed. The component to be mixed may be mixed by the mixing assembly.

[0093] In some embodiments, a mixing assembly comprises one or more inflatable chambers. The inflatable chamber(s) may also each contain internal volumes. Such internal volume(s) may contain, be capable of containing, and / or be configured to contain a fluid. Some embodiments may comprise inflating and / or deflating one or more inflatable chambers (e.g., with a fluid). Inflating an inflatable chamber may locally reduce the internal volume of a mixing chamber proximate to the inflatable chamber and / or deflating an inflatable chamber may locally increase the internal volume of a mixing chamber positioned proximate to the inflatable chamber.

[0094] In some embodiments, a flexible component is positioned between one or more components of a mixing chamber and one or more components of a mixing assembly. For instance, a flexible component may be positioned between an internal volume of a mixing chamber and an internal volume of an inflatable chamber present in a mixing assembly. In such embodiments, the flexible component may form part of the mixing chamber and / or the mixing assembly (e.g., it may form a wall of the mixing chamber and / or a wall of the mixing chamber; it may serve as a wall that separates the internal volume of the mixing chamber from an internal volume of an inflatable chamber) and / or may be a component that is distinct from the mixing chamber and the mixing assembly. As one example of the latter scenario, in some embodiments, a flexible component is positioned between an external wall of the

[0095] 8

[0096] #14426357vl mixing chamber and an external wall of an inflatable chamber present in the mixing assembly. The flexible component may be integrated with the mixing chamber and / or the mixing assembly, or it may be capable of being mechanically separable from either or both such components (e.g., it may be capable of being separated from either or both components without the use of specialized tools).

[0097] Flexible components described herein, whether part of a mixing chamber and / or a mixing assembly or part of neither, may be mechanically coupled to a mixing chamber, a mixing assembly, and / or an inflatable chamber positioned in a mixing assembly. This mechanical coupling may be effectuated through the mechanical coupling of a deformable wall of such a component (e.g., a deformable wall of a mixing chamber, a deformable wall of a mixing chamber, a deformable wall of an inflatable chamber) to the flexible component.

[0098] Similarly, flexible components described herein, whether part of a mixing chamber and / or a mixing assembly or part of neither, may be mechanically coupled to an internal volume of a mixing chamber and / or to an internal volume of an inflatable chamber positioned in a mixing assembly. As noted above, in some embodiments, this may be due to the flexible component being positioned directly between such internal volumes and / or forming a wall that separates such internal volumes.

[0099] It is also possible for the mixing chamber and / or an inflatable chamber (and / or their internal volumes) to be enclosed partially or fully by walls that are deformable (and / or flexible) but are not the flexible component. In such embodiments, a change in the internal volume and / or shape of the internal volume of an inflatable chamber may cause deformation of such a wall enclosing the internal volume of the inflatable chamber, causing deformation of the flexible component, and / or deformation of the flexible component may cause deformation of such a wall enclosing the internal volume of the mixing chamber.

[0100] FIG. 1 shows one non-limiting example of a system 100 comprising a mixing chamber 102, a mixing assembly 104, and a flexible component 106. As can be seen in FIG. 1, in some embodiments, the flexible component is positioned directly between the mixing chamber and the mixing assembly. As shown in FIG. 1, in some embodiments, a mixing chamber is positioned above a mixing assembly. It is also possible for a mixing chamber to be positioned below and / or beside a mixing assembly.

[0101] As used herein, when a system component is referred to as being “on” or “adjacent” another system component, it can be directly on or adjacent the system component, or an intervening system component also may be present. A system component that is “directly on,” “directly adjacent,” or “in contact with” another system component means that no

[0102] 9

[0103] #14426357vl intervening system component is present. Similarly, when a system component is referred to as being “between” two other system components, it can be directly between the other two system components, or an intervening system component also may be present between the relevant system component and either or both other two system components. A system component that is “directly between” two other system components means that no intervening system components are present between that system component and the two other system components.

[0104] FIG. 2 shows another non-limiting example of a system 200 comprising a mixing chamber 202, a mixing assembly 204, and a flexible component 206. In the system shown in FIG. 2, the mixing chamber 202 contains an internal volume 208 and the mixing assembly 204 comprises an inflatable chamber 210 containing an internal volume 212. In some embodiments, like the embodiment shown in FIG. 2, a mixing assembly includes exactly one inflatable chamber. It is also possible for a mixing assembly to comprise a plurality of inflatable chambers, each containing an internal volume. This is shown schematically in FIG. 3, in which the system 300 comprises a mixing chamber 302 containing an internal volume 308, a mixing assembly 304 comprising the inflatable chambers 210A-210C, each containing one of internal volumes 212A-212C, and a flexible component 306. In embodiments including a plurality of inflatable chambers, a flexible component may be positioned between an internal volume of a mixing chamber and some or all of the internal volumes of the inflatable chambers.

[0105] As noted above, in some embodiments, a flexible component is part of a mixing chamber and / or part of a mixing assembly. In some such embodiments, the flexible component forms part of a wall of a mixing chamber and / or part of a wall of an inflatable chamber. FIG. 4 shows one non-limiting example of a system 400 in which a flexible component 406 is positioned directly between the internal volume 408 of a mixing chamber 402 and the internal volume 412 of an inflatable chamber 410 forming part of a mixing assembly 404. In FIG. 4, the flexible component 406 forms part of one of the walls enclosing the internal volume 408 and the entirety of one of the walls enclosing the inflatable chamber 410.

[0106] It should be understood that FIGs. 1-4 are exemplary and some systems described herein may both be similar in some ways to and differ in one or more ways from one or more of the systems shown in these Figures. For instance, in some embodiments, a flexible component forms part of one of the walls enclosing an internal volume of a mixing chamber but does not form part of any wall enclosing an internal volume of any inflatable chambers.

[0107] 10

[0108] #14426357vl As another example, in some embodiments, a flexible component forms the entirety of one of the walls enclosing an internal volume of an inflatable chamber but does not form part of any wall enclosing an internal volume of a mixing chamber. Similarly, a flexible component may form all of one or more of the walls enclosing an internal volume of a mixing chamber and / or may form a part of one or more of the walls enclosing an internal volume of an inflatable chamber. As yet another example, it is also possible for a flexible component to form part or all of one of the walls enclosing a first inflatable chamber and form part or all of one of the walls enclosing a second inflatable chamber (in addition to, or as an alternative to, forming part or all of one of the walls enclosing a mixing chamber). It is also possible for a flexible component to form part or all of two or more (or all walls) enclosing one or more inflatable chambers and / or one or more mixing chambers.

[0109] It is also possible for a system to comprise two flexible components. In some such embodiments, each may be positioned on a different side of an internal volume of a mixing chamber. In such embodiments, the mixing assembly may also be positioned on two or more sides of the internal volume of the mixing chamber. FIG. 5 shows one such design schematically. In FIG. 5, the system 500 comprises a mixing chamber 502 containing an internal volume 508. In the system 500, the two flexible components 506A and 506B are positioned between the mixing chamber 502 (and its internal volume 508) and the mixing assembly. The mixing assembly shown in FIG. 5 comprises two portions: the portion 504A (positioned on a side of the flexible component 506A opposite the mixing chamber 502) and the portion 504B (positioned on a side of the flexible component 506B opposite the mixing chamber 502).

[0110] It should be understood that a system may comprise two flexible components that are positioned on opposing sides of a mixing chamber (e.g., like in the embodiment shown in FIG. 5) and / or may comprise two flexible components that are positioned on adjacent sides of a mixing chamber. Additionally, some systems may comprise two flexible components that are positioned on the same side of a mixing chamber.

[0111] It should also be understood that, when a system comprises two or more flexible components, the flexible components, mixing assemblies, and / or mixing chambers may have some or more features of the designs shown in FIGs. 1-4. For instance, one or more flexible components may make up part or all of one of the walls enclosing an internal volume of a mixing chamber and / or one or more flexible components may make up part or all of one of the walls enclosing an internal volume of one or more inflatable chambers.

[0112] 11

[0113] #14426357vl In some embodiments, like the embodiments shown in FIGs. 1-5, a mixing assembly comprises one or more inflatable chambers that have internal volumes positioned external to the internal volume of a mixing chamber. Advantageously, positioning inflatable chambers in this manner may allow for them to be employed to effectuate mixing in the mixing chamber without causing contamination thereof by any components present in the mixing chamber.

[0114] As used herein, an “internal volume” of a system component refers to a volume of that system component that is enclosed by walls of the system component such that it can be filled with a fluid and such that this fluid can be contained therein by such walls. For instance, with reference to FIG. 6, the system component 614 comprises an internal volume 616 contained by the walls 618A-E. As shown in FIG. 6, it is possible for an internal volume to be partially, but not fully, bounded by a plurality of walls. In other words, it may be contained by a plurality of walls but still be in fluidic communication and / or contact with an environment external to thereto. It is also possible for an internal volume to be fully bounded by a plurality of walls.

[0115] As used herein, a system component has an internal volume that is “external” to the internal volume of a different system component if its internal volume is separated from that internal volume by a wall that encloses that internal volume.

[0116] For instance, with reference to FIG. 7, the system component 720 has an internal volume 722 that is not external to the internal volume 716 of the system component 714 because the internal volume 722 is not separated from the internal volume 716 by any of the walls enclosing the internal volume 716.

[0117] By contrast, with reference to FIG. 8, the system component 824 has an internal volume 826 that is external to the system component’s 814 internal volume 816 because it is separated from the internal volume 816 by the wall 818B. As can be seen from FIG. 8, the system component 824 protrudes upwards and into the system component 814 by deforming a portion of the wall 818B enclosing the internal volume 816. It is therefore external to the internal volume 816.

[0118] In some embodiments, a flexible component is positioned such that inflation of an inflatable chamber causes local deformation of the flexible component towards the mixing chamber, thereby locally reducing the internal volume of the mixing chamber. Additionally or alternatively, it is also possible for a flexible component to be positioned such that deflation of an inflatable chamber causes local deformation of the flexible component away from the mixing chamber, thereby locally increasing the internal volume of the mixing 12

[0119] #14426357vl chamber. Deformation of components described herein (e.g., flexible components, deformable walls) may comprise changing a position of the component, may comprise changing an orientation of a component, may comprise changing a shape of a component, and / or may comprise changing a surface area of one or more walls of the component. For instance, deformation of a component may comprise raising or lowering the deformable component, changing the concavity of a portion of the component, and / or stretching or shrinking the component.

[0120] One example of such inflation and deflation, and the associated deformation, is shown schematically in FIG. 9. The top panel of FIG. 9 shows a system 900 comprising a mixing chamber 902, a mixing assembly 904, and a flexible component 906. The mixing chamber 902 has an internal volume 908. The mixing assembly 904 comprises an inflatable chamber 910 having an internal volume 912. The middle panel of FIG. 9 shows this system after inflation of the inflatable chamber 910. This inflation increases the internal volume 912, which causes local deformation of the flexible component 906 towards the mixing chamber 902 and thereby locally reduces the internal volume 908 of the mixing chamber 902. The bottom panel of FIG. 9 shows this system after deflation of the inflatable chamber 910. This deflation reduces the internal volume 912, which causes local deformation of the flexible component 906 away from the mixing chamber 902 and thereby locally increases the internal volume 908 of the mixing chamber 902.

[0121] In some embodiments, like the embodiment shown in FIG. 9, locally increasing the internal volume of a mixing chamber increases the total volume of the mixing chamber and / or locally reducing the internal volume of a mixing chamber. It is also possible for a mixing chamber to be subject to one or more local increases in internal volume and one or more local decreases in internal volume having magnitudes such that the total internal volume of the mixing chamber is unchanged. This may be caused by, for example, employing a mixing assembly comprising two mixing chambers, one of which is inflated by an amount that the other is deflated.

[0122] Additionally, like the embodiment shown in FIG. 9, locally deforming a flexible component may comprise deforming the entirety of the flexible component. It is also possible for local deformation of a flexible component to comprise deforming only a portion thereof. The flexible component may comprise one or more further portions that are undeformed during such local deformation and / or that are deformed differently (e.g., in a different direction and / or with a different magnitude) during such local deformation.

[0123] 13

[0124] #14426357vl In some embodiments, methods are provided. The methods described herein may comprise employing one or more of the systems described herein and / or one or more components present in a system described herein. Some exemplary methods are described in further detail below.

[0125] FIG. 10 shows one non-limiting example of a method 1028. Methods described herein may comprise performing one or more of the steps shown in FIG. 10. Such steps may be performed simultaneously and / or sequentially. The step 1030 shown in FIG. 10 comprises inflating a first inflatable chamber, thereby locally deforming a first flexible component towards a mixing chamber and locally reducing the internal volume of the mixing chamber. The step 1032 shown in FIG. 10 comprises inflating a second inflatable chamber, thereby locally deforming a second flexible component towards the mixing chamber and locally reducing the internal volume of the mixing chamber. The step 1034 shown in FIG. 10 comprises deflating the first inflatable chamber, thereby locally deforming the first flexible component away from the mixing chamber and locally reducing the internal volume of the mixing chamber. The step 1036 shown in FIG. 10 comprises deflating the second inflatable chamber, thereby locally deforming the second flexible component away from the mixing chamber and locally reducing the internal volume of the mixing chamber.

[0126] In some embodiments, a method comprises inflating and deflating a single inflatable chamber. This could be accomplished by performing the method steps 1030 and 1034 (in either order and / or in a repeating cycle). In some embodiments, a method comprises inflating and deflating two or more inflatable chambers. This could be accomplished by performing all of the method steps 1030-1036 shown in FIG. 10 (in any order and / or in a repeating cycle in which these steps are performed in any order). When multiple inflatable chambers are inflated and deflated, the timing of the inflation and deflation may be such that two chambers are inflated during periods of time that overlap, such that two chambers are deflated during periods of time that overlap, and / or such that one chamber is inflated while another chamber is being deflated.

[0127] For all methods, the timing and rate of any inflation and deflation of an inflatable chamber may be selected as desired. For instance, in some embodiments in which two inflatable chambers are inflated, one inflatable chamber (e.g., a first inflatable chamber) is inflated before another inflatable chamber (e.g., a second inflatable chamber) is inflated. As another example, in some embodiments in which two inflatable chambers are deflated, one inflatable chamber (e.g., a first inflatable chamber) is deflated before another inflatable chamber (e.g., a second inflatable chamber) is deflated. As a third example, in some

[0128] 14

[0129] #14426357vl embodiments, one inflatable chamber (e.g., a first inflatable chamber) is inflated while another inflatable chamber (e.g., a second inflatable chamber) is deflated. This may cause the internal volume of the mixing chamber, as a whole, to be substantially unchanged (e.g., if the rate of inflation of the first inflatable chamber matches the rate of deflation of the second inflatable chamber).

[0130] It should also be noted that some methods may comprise inflating more than two inflatable chambers and / or deflating more than two inflatable chambers.

[0131] When two or more inflatable chambers are present, such inflatable chambers may be capable of being inflated and / or deflated independently from each other and / or may be configured to be inflated and / or deflated independently from each other. It is also possible for a method to comprise inflating and deflating two or more inflatable chambers in a predetermined pattern (and / or for two or more inflatable chambers to be capable of being inflated and / or deflated in a pre-determined pattern and / or for two or more inflatable chambers to be configured to be inflated and / or deflated in a pre-determined pattern). A variety of suitable patterns may be employed and a variety of suitable types of mixing may be performed. In some embodiments, a pre-determined pattern causes and / or a mixing effectuated during a method described herein causes wave-motion mixing. In some embodiments, a pre-determined pattern causes and / or a mixing effectuated during a method described herein causes rocking-motion mixing.

[0132] As noted above, in some embodiments, a system comprises two or more flexible components. In such embodiments, inflation and / or deflation of some inflatable chambers may cause deformation of one such flexible component and inflation and / or deflation of other inflatable chambers may cause deformation of a different flexible component. It is also possible for inflation and deflation of two or more inflatable chambers to cause deformation of the same flexible component (e.g., different portions thereof). In such embodiments, the first and second flexible components shown in FIG. 10 may be the same flexible component (i.e., they may both be the first flexible component).

[0133] Inflating and deflating inflatable chambers may be accomplished in a variety of suitable manners. In some embodiments, inflating an inflatable chamber comprises filling the inflatable chamber (e.g., introducing a fluid into an internal volume positioned therein) with a fluid. In some embodiments, deflating an inflatable chamber comprises removing a fluid from the inflatable chamber (e.g., removing a fluid from an internal volume positioned therein). Filling an inflatable chamber (and / or its internal volume) with a fluid may increase the internal volume of the inflatable chamber, which may cause one or more deformable 15

[0134] #14426357vl walls therein to deform outwards (e.g., and towards a mixing chamber). Removing a fluid from an inflatable chamber (and / or its internal volume) may decrease the internal volume of the inflatable chamber, which may cause one or more deformable walls therein to deform inwards (e.g., and away from a mixing chamber).

[0135] A variety of suitable fluids may be filled into and / or removed from the inflatable chambers described herein (and / or their internal volumes), non-limiting examples of which include liquids (e.g., water) and gases (e.g., air, such as compressed air). Such fluids may be supplied to and / or removed from inflatable chambers (and / or their internal volumes) by a variety of suitable methods, one example of which is by the use of pneumatics (e.g., a fluid may be supplied and / or removed pneumatically). Supplying and / or removing a fluid pneumatically may comprise adjusting the pressure of in a pneumatic line and / or turning a pneumatic line on and / or off. Advantageously, the use of pneumatics to supply and / or remove fluid from an inflatable chamber (and / or an internal volume thereof) may allow for the inflation and deflation of inflatable chambers to be accomplished by the use of common laboratory supply lines (e.g., air supply lines, water supply lines) and / or via automated fluidflow controllers (e.g., fluid-flow controllers that control the flow of fluid through tubing).

[0136] Another example of a manner in which a fluid may be supplied to and / or removed from the inflatable chambers described herein (and / or their internal volumes) is via a syringe and / or a syringe pump. For instance, a barrel of a syringe may be filled with a such a fluid, and this fluid may be expelled from the syringe into an inflatable chamber (and / or its internal volume). The fluid may be expelled from the syringe by depressing a plunger positioned within the barrel, which may be accomplished via a syringe pump mechanically coupled to the plunger. As another example, a fluid may be removed from an inflatable chamber described herein (and / or its internal volume) by drawing it into a barrel of a syringe. In such embodiments, the pressure in the barrel may be reduced by raising a plunger positioned therein, which may draw the fluid from the inflatable chamber (and / or its internal volume) into the barrel. The plunger may be raised by a syringe pump mechanically coupled thereto.

[0137] Plungers positioned within syringe barrels may be mechanically coupled to syringe pumps in a variety of suitable manners, such as via a snap fitting, a pressure fitting, and / or an adhesive.

[0138] In some embodiments, inflation and / or deflation of one or more inflatable chambers may cause mixing of a component present in a mixing chamber (e.g., in an internal volume therein). In some embodiments, a liquid is present in an internal volume of a mixing chamber, and inflating and / or deflating one or more inflatable chambers causing mixing of 16

[0139] #14426357vl the liquid. The mixing may be due to the mechanical agitation of the component present in the mixing chambers by the inflatable chamber(s) (e.g., through the flexible component and / or a wall of the mixing chamber to which the flexible component is adjacent, such as a bottom wall).

[0140] The inflatable chambers described herein may have a variety of suitable designs. In some embodiments, an inflatable chamber takes the form of a container that may be filled with a fluid (e.g., that contains an internal volume that may be filled with a fluid). As noted above, the container may have one or more deformable walls (e.g., a wall that is also a flexible component as described elsewhere herein and / or a deformable wall that is mechanically coupled to such a flexible component). In some embodiments, an inflatable chamber takes the form of a plurality of deformable walls that contain an internal volume (e.g., on all sides) and / or takes the form of a flexible bag that contains an internal volume.

[0141] The internal volumes of inflatable chambers may be selected as desired. In some embodiments, an inflatable chamber contains an internal volume through which fluid can readily flow in multiple directions. For instance, an inflatable chamber may comprise walls that enclose an internal volume and flow through the internal volume may be unobstructed. In some embodiments, an inflatable chamber comprises one or more components that obstruct flow through an internal volume contained therein in one or more locations and / or in one or more directions. As one example, in some embodiments, an inflatable chamber comprises one or more welds, such as one or more welds that adhere together portions of opposing walls (or opposing portions of a single wall) enclosing the internal volume.

[0142] FIG. 11 shows one non-limiting example of two inflatable chambers, each comprising a plurality of welds. In FIG. 11, two inflatable chambers, and their internal volumes, are enclosed by an upper wall, a lower wall, and welds welding together the upper and lower walls. The inflatable chambers are positioned on the same substrate, different portions of which form their lower walls. Each inflatable chamber further comprises welds that obstruct flow through their internal volumes. As also shown in FIG. 11, inflatable chambers may comprise linear welds, welds intersecting an outer edge or wall of the inflatable chamber also connecting the walls welded together, and / or multiple welds having the same design. It is also possible for an inflatable chamber to comprise welds having different designs (e.g., curved welds, two or more welds that differ from each other in one or more ways, welds that do not intersect an outer edge or wall of the inflatable chamber also connecting the walls welded together).

[0143] 17

[0144] #14426357vl In some embodiments, an inflatable chamber comprises one or more features that facilitate effective use in the systems described herein. As one example, and as also shown in FIG. 11, an inflatable chamber may be in fluidic communication with an inlet through which a fluid (e.g., air, such as compressed air) may be introduced into and / or removed from an internal volume contained therein. In some embodiments, like the embodiment shown in FIG. 11, one inlet may be employed for (and / or capable of and / or configured for) both filling an inflatable chamber with a fluid and removing a fluid from the inflatable chamber. It is also possible for a system to comprise both an inlet to be employed for (and / or capable of and / or configured for) filling an inflatable chamber with fluid and an outlet to be employed for (and / or capable of and / or configured for) removing fluid from the inflatable chamber.

[0145] As another example, and as also shown in FIG. 11, one or more welds present in an inflatable chamber may facilitate connection (e.g., mechanical connection, adhesion) with a mixing chamber. With reference to FIG. 11, a mixing chamber may be placed above or below an inflatable chamber, and a weld present in the inflatable chamber may facilitate connection with the mixing chamber.

[0146] FIG. 12 depicts another exemplary pair of inflatable chambers. Like the pair of inflatable chambers depicted in FIG. 11, the inflatable chambers depicted in FIG. 12 are positioned on the same substrate. Additionally, and also like the pair of inflatable chambers depicted in FIG. 11, the inflatable chambers depicted in FIG. 12 are each in fluidic communication with an inlet through which a fluid may be introduced into and / or removed from an internal volume contained therein. The inflatable chambers shown in FIG. 12 have internal volumes that are partially interdigitated. This may be desirable for causing particular changes in the internal volume of a mixing chamber to which such inflatable chambers are adjacent.

[0147] FIG. 13 depicts a further example of a plurality of inflatable chambers. In FIG. 13, four different inflatable chambers are positioned in the same housing. A flexible component is positioned over this housing and also partially contains the internal volumes of the inflatable chambers. Each inflatable chamber shown in FIG. 13 also includes an inlet on a side thereof through which a fluid can be introduced thereinto and / or removed therefrom.

[0148] FIG. 14 depicts yet another further example of a plurality of inflatable chambers.

[0149] This Figure depicts two bags that can be inflated (e.g., by fluid supplied via the tubing also depicted in FIG. 14).

[0150] The flexible components described herein may have a variety of suitable designs. As described elsewhere herein, some flexible components make up part or all of a wall present in

[0151] 18

[0152] #14426357vl a mixing assembly, an inflatable chamber, and / or a mixing chamber (and / or enclosing an internal volume therein). As also described elsewhere herein, some flexible components are distinct and / or mechanically separable from a mixing assembly, an inflatable chamber, and / or a mixing chamber. Such flexible components may be mechanically coupled to the mixing assembly, the inflatable chamber, and / or the mixing chamber and / or their internal volumes (e.g., via a deformable and / or flexible wall thereof).

[0153] In some embodiments, a flexible component takes the form of a layer and / or a wall comprising, consisting of, and / or consisting essentially of a flexible material. The flexible material may be sufficiently flexible to be capable of being deformed as described elsewhere herein upon the inflation and deflation of inflatable chambers. Flexible components described herein may have a variety of suitable elastic moduli. In some embodiments, a flexible component has a thickness of between 0.1 mm and 10 mm and an elastic modulus of between 0.05 MPa and 5 MPa. In some embodiments, the flexible material is an elastomer, such as silicone.

[0154] In some embodiments, a flexible component comprises a membrane, such as a gas- permeable membrane. In such embodiments, gas may be supplied to one or more system components through the gas-permeable membrane. For instance, in some embodiments, a gas-permeable membrane is positioned directly between the internal volume of a mixing chamber and the internal volume of an inflatable chamber, and gas is supplied from the inflatable chamber to the mixing chamber through the gas-permeable membrane. This may be desirable when it would be beneficial to supply a particular gas to a liquid contained in the internal volume of the mixing chamber, such as supplying oxygen to a cell-culture medium contained in the internal volume of the mixing chamber.

[0155] The mixing chambers described herein may have a variety of suitable designs. As noted elsewhere herein, some mixing chambers may comprise one or more deformable and / or flexible walls (e.g., enclosing an internal volume therein). Non-limiting examples of suitable mixing chamber designs include bags (e.g., cell-culture bags), flasks, and bioreactors (e.g., mini bioreactors). FIG. 15 shows one non-limiting example of a mixing chamber taking the form of a bag.

[0156] As noted above, mixing chambers may contain internal volumes containing fluids, such as liquids and gases. In some embodiments, one or more solids are suspended in such fluids. As one non-limiting example, in some embodiments, a mixing chamber contains a liquid in which cells are suspended.

[0157] 19

[0158] #14426357vl In some embodiments, an internal volume of a mixing chamber is in fluidic communication with a source of a gas, is capable of being in fluidic communication with a source of a gas, and / or is configured to be in fluidic communication with a source of a gas. For instance, an internal volume of a mixing chamber may be in fluidic communication with a source of air (e.g., fresh air), capable of being in fluidic communication with a source of air (e.g., fresh air), and / or configured to be in fluidic communication with a source of air (e.g., fresh air). The fluidic communication may occur in a variety of suitable manners, nonlimiting examples of which include via an open side in the fluidic chamber, via an opening in the fluidic chamber, via a port, and / or via tubing. Without wishing to be bound by any particular theory, it is believed that introducing fresh air into an internal volume of a mixing chamber may be particularly beneficial because it may allow for the gases present in fresh air (e.g., oxygen) to be supplied to a fluid present therein. This may be advantageous in comparison to supplying such an internal volume with recirculating air, because some gases present in the recirculating air that it may be desirable to supply to an internal volume of a mixing chamber may be removed during the recirculation.

[0159] The mixing chambers described herein may comprise internal volumes having a variety of suitable magnitudes. In some embodiments, a mixing chamber has an internal volume having a size of greater than or equal to 10 microliters, greater than or equal to 20 microliters, greater than or equal to 50 microliters, greater than or equal to 75 microliters, greater than or equal to 100 microliters, greater than or equal to 200 microliters, greater than or equal to 500 microliters, greater than or equal to 750 microliters, greater than or equal to 1 L, greater than or equal to 2 L, greater than or equal to 5 L, greater than or equal to 7.5 L, greater than or equal to 10 L, greater than or equal to 20 L, greater than or equal to 50 L, greater than or equal to 75 L, greater than or equal to 100 L, or greater than or equal to 200 L. In some embodiments, a mixing chamber has an internal volume having a size of less than or equal to 500 L, less than or equal to 200 L, less than or equal to 100 L, less than or equal to 75 L, less than or equal to 50 L, less than or equal to 20 L, less than or equal to 10 L, less than or equal to 7.5 L, less than or equal to 5 L, less than or equal to 2 L, less than or equal to 1 L, less than or equal to 750 microliters, less than or equal to 500 microliters, less than or equal to 200 microliters, less than or equal to 100 microliters, less than or equal to 75 microliters, less than or equal to 50 microliters, or less than or equal to 20 microliters. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 10 microliters and less than or equal to 500 L). Other ranges are also possible.

[0160] 20

[0161] #14426357vl The various system components described herein may be employed together in a variety of suitable manners. A further discussion of some exemplary combinations of such components is provided below.

[0162] In some embodiments, a system comprises a mixing chamber taking the form of a cell-culture bag that is disposed on a plurality of inflatable chambers. Various examples of systems having such a design are shown schematically in FIGs. 16-20. A schematic depiction of the various components of the system shown in FIG. 16 is further shown in FIG. 21. In the design shown in FIG. 19, two inflatable chambers taking the form of bags are welded to a single substrate. The cell-culture bag shown in FIG. 19 is not integrally connected to these inflatable chambers. In the design shown in FIG. 20, two inflatable chambers taking the form of bags are welded to and share a wall with a cell-culture bag. In some embodiments, a system is positioned on a chip (e.g., a chip having multiple layers). In such embodiments, the chip is interfaced with various components that can supply and / or remove fluids therefrom. FIG. 22 shows one example of such a chip and associated hardware.

[0163] FIG. 23 depicts an exemplary diagram of electronic and pneumatic systems that may be employed in the systems described herein.

[0164] The systems described herein may be employed for a variety of suitable applications and the methods described herein may comprise performing a variety of types of processes. In some embodiments, a method comprises employing a system described herein to mix one or more components (e.g., one or more components present in a mixing chamber, such as one or more fluids therein). For instance, a method may comprise employing a system described herein to mix chemicals, buffers, cell-culture media, and / or polyplexes for transfection and / or gene therapy. Other non-limiting examples of the uses to which systems described herein may be put include RNA in-vitro synthesis, the homogenization of post-thaw cryofrozen samples, and the delivery of cell therapy products.

[0165] EXAMPLE 1

[0166] This Example describes the use of a system described herein to effectuate mixing in a liquid contained in an internal volume of a mixing chamber.

[0167] A system having the design shown in FIG. 23 was employed.

[0168] Prior to mixing, the mixing chamber (a cell-culture bag) was prepared as follows. First, the mixing chamber was filled with 1000 mL of distilled water. Then, 2 mL of iodinepotassium solution and 5 mL of starch solution were added thereto, and the resultant solution 21

[0169] #14426357vl was mixed for 1 minute at 1100 rpm using a magnetic stir bar. Subsequently, the mixing chamber was allowed to equilibrate without any active mixing for 6 minutes. Finally, 4 mL of a sodium thiosulfate solution was injected into the mixing chamber over 60 seconds.

[0170] Mixing was accomplished by inflating and deflating two inflatable chambers. Three programs were employed: a fast mixing program, a moderate mixing program, and a gentle mixing program. For each mixing program, the inflation of each inflatable chamber was controlled pneumatically by opening and closing a valve supplying compressed air (Valve A for one and Valve B for the other). The programs employed are generally shown in FIG. 24, with further specific features shown in Table 1. FIGs. 25-26 show the anticipated inflation level of each inflatable chamber as a function of time for the various mixing programs.

[0171] Table 1. Features of Mixing Programs.

[0172] The extent of mixing over time was assessed by measuring the transparency of the mixing chamber as a function of time. For each mixing program, 3-5 replicates were performed. The results of these measurements are shown schematically in FIG. 27 for the fast mixing program, FIG. 28 for the moderate mixing program, and FIG. 29 for the gentle mixing program. The fast mixing program achieved complete mixing in 10-15 seconds with a standard deviation of 3.69 seconds. Additionally, the slope of the rising edge of the curve describing mixing as a function of time had a standard deviation of less than 1 second. The moderate mixing program achieved complete mixing in 30 seconds with a standard deviation of 4.79 seconds. Additionally, the slope of the rising edge of the curve describing mixing as a function of time had a standard deviation of 2.73 seconds. Therefore, both of these mixing programs achieved repeatable mixing performance. The gentle mixing program achieved complete mixing in 60 seconds. It is believed that the differences in mixing performance

[0173] 22

[0174] #14426357vl exhibited upon use of the gentle mixing program may be due to variations in the starting solution and / or the inability of the transparency measurements to capture mixing occurring at the bottom of the mixing chamber.

[0175] EXAMPLE 2

[0176] This Example describes the use of a system having the design shown in the top left panel of FIG. 22 to perform cell culture in the mixing chambers (i.e., in which the cell-culture chambers therein served as mixing chambers).

[0177] Four different conditions were employed: two in which neither mixing nor perfusion were performed (Al and A2), and two in which both mixing and perfusion were performed (Bl and B2). When performed, mixing was effectuated by inflating and deflating the two inflatable chambers. Perfusion, when performed, was effectuated by replacing the full volume of the cell-culture chambers over ten minutes on each of days four, five, and six of the experiment. The first day of the experiment was considered day zero, so days four, five, and six were the fifth, sixth, and seventh day of the experiment, respectively.

[0178] FIG. 30 shows the cell concentration on each day of the experiment and FIG. 31 shows the cell viability percentage on each day of the experiment. As can be seen from FIGs. 30 and 31, the performance of mixing and perfusion during cell culture resulted in increased cell growth and cell viability in comparison to conditions during which mixing and perfusion were not performed. It is believed that this occurred because the mixing and perfusion promoted uniform oxygenation and nutrient distribution. It is believed that the initial mixing to which the cell-culture chamber experiencing condition B2 was subjected may have caused an inadvertent loss of volume, and that, had this volume loss not occurred, the cell culture performed in that chamber may have had similar properties to the cell culture performed according to condition B 1.

[0179] It should also be noted that the system shown in FIG. 22 advantageously allowed for efficient washing of the cell-culture chambers by replacing the media and for uniform cell seeding within the cell-culture chamber. The latter can be particularly challenging in vessels where manual mechanical mixing via pipetting is not possible.

[0180] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications 23

[0181] #14426357vl is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0182] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0183] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0184] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, 24

[0185] #14426357vl additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0186] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0187] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0188] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0189] 25

[0190] #14426357vl

Claims

CLAIMSWhat is claimed is:

1. A system, comprising: a mixing chamber containing an internal volume; a mixing assembly comprising an inflatable chamber containing an internal volume; and a flexible component positioned between the internal volume of the mixing chamber and the internal volume of the inflatable chamber, wherein: the internal volume of the inflatable chamber is positioned external to the internal volume of the mixing chamber, the flexible component is positioned such that inflation of the inflatable chamber causes local deformation of the flexible component towards the mixing chamber, thereby locally reducing the internal volume of the mixing chamber, and the flexible component is positioned such that deflation of the inflatable chamber causes local deformation of the flexible component away from the mixing chamber, thereby locally increasing the internal volume of the mixing chamber.

2. A method, comprising: in the system of claim 1: inflating the inflatable chamber, thereby locally deforming the flexible component towards the mixing chamber and locally reducing the internal volume of the mixing chamber.

3. The method of claim 2, further comprising deflating the inflatable chamber.

4. A method, comprising: in the system of claim 1: deflating the inflatable chamber, thereby locally deforming the flexible component away from the mixing chamber and locally increasing the internal volume of the mixing chamber.26#14426357vl5. The method of claim 2, further comprising inflating a second inflatable chamber, thereby locally deforming a second flexible component towards the mixing chamber and locally reducing the internal volume of the mixing chamber.

6. The method of claim 2, further comprising deflating a second inflatable chamber, thereby locally deforming a second flexible component away from the mixing chamber and locally increasing the internal volume of the mixing chamber.

7. The method of claim 5, wherein the second flexible component is the same component as the flexible component.

8. The method of claim 5, wherein the inflatable chamber is inflated before the second inflatable chamber is inflated.

9. The method of claim 4, wherein the system further comprises a second deflatable chamber, and wherein the inflatable chamber is deflated before the second inflatable chamber is deflated.

10. The method of claim 6, wherein the inflatable chamber is inflated while the second inflatable chamber is deflated, thereby causing the internal volume of the mixing chamber, as a whole, to be substantially unchanged.

11. The system of claim 1, further comprising a second inflatable chamber, wherein the inflatable chamber and the second inflatable chamber are capable of being inflated and deflated independently from each other.

12. The method of claim 2, further comprising inflating and deflating the inflatable chamber in a pre-determined pattern.

13. The system of claim 1, wherein inflating and / or deflating the inflatable chamber causes mixing of a liquid present in the internal volume of the mixing chamber.

14. The system of claim 13, wherein the mixing comprises wave-motion mixing.27#14426357vl15. The system of claim 13, wherein the mixing comprises rocking-motion mixing.

16. The system of claim 1, wherein inflating the inflatable chamber comprises filling the inflatable chamber with a fluid.

17. The system of claim 1, wherein deflating the inflatable chamber comprises removing a fluid from the inflatable chamber.

18. The system of claim 16, wherein the fluid is water.

19. The system of claim 16, wherein the fluid is a gas.

20. The system of claim 16, wherein the fluid is supplied pneumatically.

21. The system of claim 1, wherein the inflatable chamber comprises one or more welds.

22. The system of claim 1, wherein the flexible component is positioned directly between the internal volume of the mixing chamber and the internal volume of the inflatable chamber.

23. The system of claim 1, wherein the flexible component forms a wall of the mixing chamber.

24. The system of claim 1, wherein the flexible component forms a wall of the inflatable chamber.

25. The system of claim 1, wherein the mixing chamber comprises a flexible wall, and wherein the flexible wall of the mixing chamber is mechanically coupled to the flexible component.

26. The system of claim 1, wherein the inflatable chamber comprises a flexible wall, and wherein the flexible wall of the inflatable chamber is mechanically coupled to the flexible component.

27. The system of claim 1, wherein the flexible component comprises an elastomer.28#14426357vl28. The system of any claim 27, wherein the elastomer is silicone.

29. The system of claim 1, wherein the flexible component comprises a membrane.

30. The system of claim 29, wherein the membrane is permeable to a gas.

31. The method of claim 2, further comprising supplying a gas from an inflatable chamber, through the membrane, and to a liquid present in the internal volume of the mixing chamber.

32. The system of claim 1, wherein the mixing chamber is a bag.

33. The system of claim 1, wherein the mixing chamber is a flask.

34. The system of claim 1, wherein the mixing chamber is a bioreactor.

35. The system of claim 1, wherein the mixing chamber has an internal volume having a size of greater than or equal to 10 microliters and less than or equal to 500 L.

36. The system of claim 1, wherein a liquid is present in the internal volume of the mixing chamber.

37. The system of claim 36, wherein cells are suspended in the liquid.

38. The method of claim 2, further comprising employing the system to mix chemicals, buffers, and / or cell-culture media.

39. The method of claim 2, further comprising employing the system during RNA in-vitro synthesis.

40. The method of claim 2, further comprising employing the system to mix polyplexes for transfection and / or gene therapy.29#14426357vl41. The method of claim 2, further comprising employing the system to homogenize postthaw cryofrozen samples.

42. The method of claim 2, further comprising employing the system to deliver cell therapy products.30#14426357vl

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