Cell culture dish comprising two-dimensional negligible volume bubbler

Abstract

An exemplary cell culture dish is described that includes a housing defining a cavity operable to maintain a cell culture and an open end fluidly connected to the cavity, the open end surrounded by a peripheral edge of the housing, and a bubbler coupled to the open end at the peripheral edge. The bubbler includes a membrane having a first peripheral edge portion and a plate having a second peripheral edge portion. The first peripheral edge portion is sealingly connected to the second peripheral edge portion such that the membrane is spaced from the plate by less than about 100 microns to form a fluid channel between the membrane and the plate. The membrane further includes a plurality of spaced apart pores. Methods of using the cell culture dishes are also described.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 428,815, filed on Nov. 30, 2022, under 35 U.S.C. § 119, the content of which is incorporated herein by reference in its entirety and made a part hereof. Technical field

[0003] The present disclosure relates to a method and a cell culture dish. More particularly, the present disclosure relates to a cell culture dish having a bubbler. Background art

[0004] A common method for supplying gas (e.g., air, oxygen) to a cell culture dish is to use a bubbler. Bubbles generated by the bubbler in the liquid medium in the dish help to stir and oxygenate the liquid medium (and the cells therein) in the cell culture dish. Generally, the bubbler is a pipe that extends into the volume of the dish, and the pipe is configured with orifices through which gas escapes from the pipe. However, the relatively large cross - sectional area of these pipes and / or associated manifold devices may allow the backflow of the liquid medium in the pipe or the retention of the liquid medium in the pipe. Additionally, such large three - dimensional structures may require complex manufacturing steps, excessive manufacturing materials, and may provide limited flexibility, thus being prone to damage during transportation and / or use in flexible vessels such as bags. Summary of the invention

[0005] Exemplary embodiments of the present disclosure provide a cell culture dish. The cell culture dish may include a housing that defines a cavity operable to maintain a cell culture and an open end fluidly connected to the cavity, the open end being surrounded by a peripheral edge of the housing. The cell culture dish may include a bubbler coupled to the open end at the peripheral edge, the bubbler including a membrane and a plate, the membrane including a first peripheral edge portion, the plate including a second peripheral edge portion, the first peripheral edge portion being sealingly connected to the second peripheral edge portion such that the membrane is spaced from the plate by less than about 100 microns to form a fluid channel therebetween, the membrane further including a plurality of spaced - apart holes extending through the thickness of the membrane, the fluid channel being in fluid communication with the cavity through the plurality of holes.

[0006] In some embodiments, the thickness of the membrane can be less than about 2.5 millimeters. In some embodiments, the membrane can comprise a polymer. In some embodiments, the diameter of each of the plurality of pores can be less than about 1 millimeter. In some embodiments, the cell culture dish can further comprise a gas line in fluid communication with the fluid channel. In some embodiments, the cell culture dish can further comprise a check valve that is operable to allow fluid to flow into the fluid channel in a first direction and prevent fluid from flowing out of the fluid channel in a second direction. In some embodiments, the check valve is located in the gas line. In some embodiments, the gas line is attached in a fixed manner between the membrane and the plate. In some embodiments, the membrane can include a first major surface facing the chamber, and the first major surface can be non-planar. In some embodiments, the first major surface can be a treated surface configured to enhance and / or inhibit cell attachment to the first major surface. In some embodiments, the treated first major surface can include a net negative surface charge. In some embodiments, the treated first major surface can include a coating. In some embodiments, the wall of the housing can be flexible. In some embodiments, the membrane and the plate can be sealingly connected to each other by at least one of an airtight adhesive, heat sealing, hot melting, ultrasonic welding, or solvent bonding.

[0007] Exemplary embodiments of the present disclosure provide a method of bubbling a cell culture. The method can comprise depositing the cell culture into a cell culture dish comprising: a housing defining a chamber operable to maintain the cell culture and an open end fluidly connected to the chamber, the open end being surrounded by a peripheral edge of the housing; and a bubbler coupled to the open end at the peripheral edge, the bubbler comprising a membrane and a plate, the membrane comprising a first peripheral edge portion, the plate comprising a second peripheral edge portion, the second peripheral edge portion being sealingly connected to the first peripheral edge portion such that the membrane is spaced apart from the plate by less than about 100 μm to form a fluid channel between the membrane and the plate, the membrane further comprising a plurality of spaced-apart pores extending through the thickness of the membrane, the fluid channel being in fluid communication with the chamber through the plurality of pores. The method can further comprise flowing a gas into the fluid channel, the gas flowing out of the plurality of pores into the housing.

[0008] In some embodiments, the diameter of each of the plurality of holes can be less than about 1000 microns. In some embodiments, the gas can flow through a gas line extending between the fluid channel and a gas source. In some embodiments, the gas can flow through a one-way valve and then enter the fluid channel. In some embodiments, the gas can be a first gas, and the method further includes flowing a second gas different from the first gas into the chamber. In some embodiments, the membrane can include a first major surface facing the chamber, and the first major surface is treated to enhance and / or inhibit cell attachment to the first major surface. In some embodiments, the deposition of the cell culture can include depositing an adherent cell culture and a suspension cell culture.

[0009] Exemplary embodiments of the present disclosure provide a method of forming a cell culture bubbler. The method can include: positioning a membrane opposite a plate including a second peripheral edge portion, the membrane including a first peripheral edge portion and a plurality of holes extending through the thickness of the membrane; and sealingly connecting the first peripheral edge portion to the second peripheral edge portion such that the membrane is spaced apart from the plate by a distance less than about 100 microns to form a fluid channel between the membrane and the plate.

[0010] In some embodiments, the sealing can include at least one of an airtight adhesive, a heat seal, a hot melt, an ultrasonic weld, or a solvent bond. In some embodiments, the method can further include treating the surface of the membrane to enhance and / or inhibit cell attachment to the surface. In some embodiments, the treatment can include a corona gas treatment. In some embodiments, the treated surface can include a net negative charge after the treatment. In some embodiments, the treatment can include applying a coating to the surface. In some embodiments, the plurality of holes can be formed by laser ablation. In some embodiments, the membrane can include a first major surface facing the fluid channel and a second major surface opposite the first major surface, and the second major surface includes one or more protrusions extending therefrom. In some embodiments, the membrane can include a first major surface facing the fluid channel and a second major surface opposite the first major surface, and the second major surface can be non-planar.

[0011] Embodiments of the present disclosure will be described below. However, the present disclosure is not limited to the described embodiments, and various modifications of the present disclosure are possible without departing from the basic principles described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Various embodiments are disclosed by way of example only with reference to the following accompanying schematic diagrams, in which corresponding reference numerals indicate corresponding parts. Identical reference numerals on different schematic views identify the same or functionally similar structural elements.

[0013] Figure 1 is a cross-sectional view of an exemplary cell vessel suitable for practicing exemplary embodiments of the present disclosure;

[0014] Figure 2 is another cross-sectional view of an exemplary cell vessel suitable for practicing exemplary embodiments of the present disclosure;

[0015] Figures 3A - 3F is a top view of an exemplary membrane suitable for practicing exemplary embodiments of the present disclosure;

[0016] Figures 4A - 4C is a cross-sectional view of an exemplary membrane suitable for practicing exemplary embodiments of the present disclosure; and

[0017] Figures 5A - 5C is a top view of an element of an exemplary membrane suitable for practicing exemplary embodiments of the present disclosure. DETAILED DESCRIPTION

[0018] The present disclosure is not limited to the specific methods, materials, and modifications described, and thus, these methods, materials, and modifications may vary. Additionally, the terms used herein are for the purpose of describing particular aspects only and are not intended to limit the scope of the claims. The claims are also not limited to the disclosed aspects.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Any method, apparatus, or material similar or equivalent to those described herein may be used to practice or test the exemplary embodiments.

[0020] As used herein, the term "substantially" is synonymous with terms such as "near", "very near", "about", "substantially", "approximately", "approximates to", "close to", "essentially", "nearby", "in the vicinity of", etc., and these terms may be used interchangeably in the specification and claims. The term "near" is synonymous with terms such as "nearby", "close to", "adjacent to", "adjoining", "immediately adjacent to", "abutting", etc., and these terms may be used interchangeably in the specification and claims. The term "substantially" is intended to denote a value within ten percent of the specified value.

[0021] Unless otherwise specified, in this application, the use of "or" is with respect to a "non-exclusive" arrangement. For example, when stating "item x is A or B", it should be understood that this can mean one of the following: (1) item x is only one or the other of A and B; (2) item x is both A and B. In other words, the word "or" is not used to define an "exclusive or" arrangement. For example, an "exclusive or" arrangement of the statement "item x is A or B" would require that x can only be one of A and B. Further, as used herein, "and / or" means a grammatical conjunction used to indicate that one or more of the recited elements or conditions may be included or occur. For example, a device including a first element, a second element, and / or a third element is intended to be understood as any of the following structural arrangements: a device including the first element; a device including the second element; a device including the third element; a device including the first and second elements; a device including the first and third elements; a device including the first, second, and third elements; or, a device including the second and third elements.

[0022] As used herein, the phrases "comprises at least one of" and "comprising at least one of" in connection with a system or element mean that the system or element includes one or more of the elements listed after the phrase. For example, a device including at least one of a first element, a second element, and a third element is intended to be understood as any of the following structural arrangements: a device including the first element; a device including the second element; a device including the third element; a device including the first and second elements; a device including the first and third elements; a device including the first, second, and third elements; or, a device including the second and third elements. When the phrase "used in at least one of the following:" is used herein, it is intended to be interpreted in a similar manner.

[0023] Embodiments of the present disclosure include a cell culture dish having a bubbler that is operable to avoid using a three-dimensional (3D) air supply channel to supply air or gas to the bubble emitter holes of the bubbler. Embodiments of the present disclosure provide a two-dimensional (2D) membrane that advantageously reduces the volume occupied by the bubbler within the dish or, more specifically, within the cell culture dish. Embodiments of the present disclosure are operable to reduce the amount of liquid and cell retention in the air manifold device of the bubbler that can enter below the bubble emitter holes. Further, embodiments of the present disclosure allow for a lower cost of manufacture compared to traditional bubblers. Exemplary embodiments reduce the number of manufacturing steps to produce the bubbler and reduce the amount of material required to manufacture and maintain the flexibility of the bubbler. Accordingly, the embodiments reduce the likelihood of breakage and damage during transportation and use in flexible vessels such as bags.

[0024] ReferenceFigure 1 and 2 , showing a cross-sectional view of an exemplary cell culture dish 100 suitable for practicing embodiments of the present disclosure. The cell culture dish 100 includes a housing 102 and a bubbler 104. The housing 102 is operable to maintain a cell culture and a fluid. The housing 102 defines a cavity 106 and has an open end 108 fluidly connected to the cavity 106. Embodiments of the housing 102 may be made of a solid material or a flexible material. In some embodiments, the housing 102 may be made of a transparent material or an opaque material. In some embodiments, the housing 102 may be made of a glass material or a flexible plastic or polymer material such that the housing 102 is operable to expand in response to air or gas moving into the housing 102 and contract in response to air or gas moving out of the housing 102. The open end 108 of the housing 102 is surrounded by a peripheral edge 110 of the housing 102. Embodiments of the peripheral edge 110 include the peripheral edge 110 being coextensive throughout its extent such that a planar object will be able to contact the entire extent of the peripheral edge 110 simultaneously.

[0025] The bubbler 104 may be coupled to the open end 108 at the peripheral edge 110 of the housing 102. The bubbler 104 may be sealingly connected to the housing 102 by an airtight seal between the membrane 112 and the housing 102, which airtight seal prevents fluid (e.g., gas, air) from passing between the housing 102 and the bubbler 104. The bubbler 104 includes a membrane 112 having a first peripheral edge portion 114 and a plate 116 having a second peripheral edge portion 118. The first peripheral edge portion 114 is the outer edge of the membrane 112 facing the plate 116 and away from the housing 102 and the peripheral edge 110. The second peripheral edge portion 118 is the outer edge of the plate 116 facing the membrane 112. The first peripheral edge portion 114 of the membrane 112 may be sealingly connected to the second peripheral edge portion 118 such that the remaining portions of the membrane 112 and the plate 116 are not sealingly connected to each other. In other words, the portions of the surfaces of the membrane 112 and the plate 116 that face each other and are not sealingly connected to each other are spaced apart, thereby forming a fluid channel 120 between the membrane 112 and the plate 116. Embodiments of the membrane 112 and the plate 116 include the membrane 112 and the plate 116 being sealingly connected to each other by at least one of an airtight adhesive, heat sealing, heat melting, ultrasonic welding, or solvent bonding. The seal between the membrane 112 and the plate 116 may be a leak-proof seal that prevents fluid, including gas, from passing between the membrane 112 and the plate 116 along the portions sealed together.

[0026] Embodiments of the membrane 112 may have the following thicknesses: (i) less than about 2.5 millimeters, (ii) between about 0.005 and about 0.025 inches, (iii) between about 0.025 and about 0.05 inches, or (iv) between about 0.05 and about 0.1 inches. In an embodiment, even when gas or air moves through the fluid channel 120 between the membrane 112 and the plate 116, the membrane 112 may not substantially extend into the cavity 106 beyond the peripheral edge 110 of the housing 102. Substantially extending into the cavity 106 would include the membrane 112 crossing a threshold of the cavity 106 defined by a plane formed by the peripheral edge 110 of the housing 102, to a degree greater than the thickness of the membrane 112. In an embodiment, the membrane 112 does not extend into the cavity 106 more than (i) greater than about 1 micron, (ii) greater than about 5 microns, (iii) greater than about 10 microns, (iv) greater than about 25 microns, (v) greater than about 50 microns, or (vi) greater than about 100 microns. In an embodiment, the membrane 112 may be spaced from the plate 116 by less than about 100 microns. In other embodiments, the membrane 112 may be spaced from the plate 116 by (i) less than about 5 microns, (ii) between about 5 and about 10 microns, (iii) between about 10 and about 25 microns, (iv) between about 25 and about 50 microns, or (vi) between about 50 and about 100 microns.

[0027] Embodiments of the membrane 112 may be made of a polymer. Other exemplary embodiments of the membrane 112 may be made of polystyrene (PS), polycarbonate (PC), polyethylene terephthalate (PET), polypropylene (PP), polyvinyl chloride (PVC), or polyetheretherketone (PEEK). Embodiments of the membrane 112 may be rigid, flexible, or semi-flexible, depending on the characteristics of the membrane 112, such as thickness, composition, and processing during manufacture.

[0028] The membrane 112 includes a plurality of spaced-apart holes 122 extending through the thickness of the membrane 112. Each of the plurality of holes 122 fluidly connects the cavity 106 with the fluid channel 120 and is operable to allow gas (e.g., air) to pass therethrough. Embodiments of the plurality of holes 122 include each hole having at least one of the following diameters: (i) between about 50 microns and about 150 microns, (ii) between about 150 microns and about 250 microns, (iii) between about 250 microns and about 500 microns, (iv) between about 500 microns and about 1 millimeter, or (v) less than about 1 millimeter. Embodiments of the plurality of holes 122 include the holes having diameters less than about 100 microns and greater than about 100 microns.

[0029] The plurality of holes 122 may be positioned in the membrane 112 in a variety of different shapes and arrangements. Refer to Figures 3A - 3F, showing some non-limiting embodiments of an exemplary arrangement of a plurality of holes 122 as shown in a top view of the membrane 112. In an embodiment, the plurality of holes 122 may be arranged in a square or rectangular pattern ( Figure 3A as shown in). In an embodiment, the plurality of holes 122 may be arranged in an annular pattern ( Figure 3B as shown in). In an embodiment, the plurality of holes 122 may be arranged in a circular or oval pattern ( Figure 3C as shown in). In an embodiment, the plurality of holes 122 may be arranged in a hexagonal pattern ( Figure 3D as shown in). In an embodiment, the plurality of holes 122 may be arranged in a triangular pattern ( Figure 3E as shown in). In an embodiment, the plurality of holes 122 may be arranged in spaced-apart groups or islands having two or more holes associated with each grouping ( Figure 3F as shown in). It should be understood that the embodiments include any type of arrangement of the plurality of holes 122 on the membrane 112, including using holes 122 having different diameters within the same membrane 112 and using different spacings between the holes 122. In other words, the embodiments include the plurality of holes 122 having different densities of holes along the membrane 112 such that the holes 122 on a first portion of the membrane 112 may be spaced apart from each other by a first distance while the holes 122 on a second portion of the membrane 112 may be spaced apart by a second distance or a varying distance less than or greater than the first distance. For example, in Figure 3E , the embodiments include that the holes 122 in portion 302 of the membrane 112 have a larger diameter and are spaced farther apart from each other compared to in portion 304 of the membrane 112 where the holes 122 have a smaller diameter and are closer together.

[0030] Embodiments of the membrane 112 include a surface 124 of the membrane 112 facing the chamber 106, which surface has been treated with a treatment or a material coating before being assembled into the cell culture dish 100. Embodiments of exemplary material coatings or treatments applied to the surface 124 are operable to inhibit or prevent cell attachment to the surface 124 of the membrane 112. In an embodiment, the surface 124 may be treated by charging the surface 124 such that it has a net negative surface charge. In an embodiment, the surface 124 may be treated such that it has a net positive surface charge. In an embodiment, the surface 124 of the membrane 112 may be treated with a corona gas treatment. Embodiments of the membrane 112 include at least one of the surface 124 or a surface 126 facing the plate 116 that has been treated with a material coating. In an embodiment, the cell culture dish 100 may include a membrane 112, wherein the surface 124 is selectively treated with a tissue culture treatment or a CellBIND surface treatment that is operable to increase the likelihood of cell attachment.

[0031] An embodiment of the cell culture dish 100 further includes a gas line 128 fluidly connected to the fluid channel 120. Embodiments of the gas line 128 allow gas to pass through the gas line 128 into the fluid channel 120 and through the plurality of holes 122. Embodiments may include a plurality of gas lines 128 fluidly connected to the fluid channel 120. In this regard, embodiments may include gas (e.g., oxygen) or air from a plurality of different gas lines 128 being provided to the fluid channel 120 and the chamber 106. Embodiments may further include a one-way valve 130 located on the gas line 128, the one-way valve being operable to allow fluid to flow into the fluid channel 120 in a first direction 134 (i.e., the direction from the gas line 128 into the fluid channel 120) and to prevent fluid from flowing in a second direction (i.e., from the fluid channel to the gas line 128). In an embodiment, the gas line 128 may be attached in a fixed manner between the membrane 112 and the plate 116 such that the gas line 128 can supply gas to the fluid channel 120. The gas line 128 is operable to removably attach to a gas source that maintains gas or air under pressure and is operable to selectively release gas or air from the gas source.

[0032] Embodiments of the membrane 112 may have a surface 124 of the membrane 112 that is planar. In an embodiment, the surface 124 of the membrane 112 may be non-planar. Refer Figures 4A - 4C , a cross-sectional view showing an exemplary embodiment of the membrane 112. In Figures 4A - 4C the embodiment shown, the membrane 112 includes a surface 124 having protrusions 404, while the opposite surface (i.e., the surface of the membrane 112 facing the plate 116) does not have protrusions 404, 406. However, embodiments include both surfaces 124, 126 of the membrane 12 having a plurality of protrusions 404, 406. As Figure 4A shown, the surface 124 and / or the surface 126 may generally be planar and flat. As Figure 4B shown, the surface 124 may include a plurality of channels 402 separated from each other by a plurality of protrusions 404. In Figure 4B the embodiment shown, the channels 402 and the protrusions 404 may have sharp edges at the top of the protrusions 404 and the bottom of the channels 402. As Figure 4C shown, the surface 124 may include a plurality of protrusions 406 and channels 408. In Figure 4C the embodiment shown, the protrusions 406 and the channels 408 may include curved edges at their respective high and low points.

[0033] Embodiments of the membrane 112 include protrusions 404, 406 that can form or define any type of shape or geometry along the surface 124 of the membrane 112 to accommodate a desired use in the cell culture dish 100. For example, it may be desirable to agitate the fluid in the chamber 106 in a counterclockwise manner about the center of the membrane 112. In this example, the channels 402, 408 and corresponding protrusions 404, 406 may be arranged in a spiral pattern, as Figure 5A shown. Figure 5B and 5C show non-limiting exemplary arrangements of the channels 402, 408 and protrusions 404, 406 on the surface 124 of the membrane 112. Figure 5B shows a plurality of crescent-shaped protrusions 404, 406. Figure 5C shows a plurality of protrusions 404, 406 presented as lines parallel to each other. Embodiments of the channels 402, 408 and protrusions 404, 406 can be arranged in any shape or design to accommodate the user's desired use. In the embodiment shown in Figures 5A - 5C , the protrusions 404, 406 are depicted by lines and the channels 402, 408 are represented by the spaces between the lines.

[0034] In practice, prior to assembling the cell culture dish 100, the surface 124 of the membrane 112 can be treated by surface treatment, and a plurality of holes 122 can be formed in the membrane 112 by, for example, laser ablation. Embodiments include surface treatment occurring before or after forming the plurality of holes 122 in the membrane 112. Advantageously, the surface treatment can be applied to the surface 124 after forming the plurality of holes 122 in the membrane 112 such that the surface treatment can penetrate the membrane 112 along the edges of the plurality of holes 122. The cell culture dish 100 can then be formed by positioning the membrane 112 opposite the plate 116 and sealingly connecting a first peripheral edge portion 114 of the membrane 112 to a second peripheral edge portion 118 of the plate 116 such that the membrane 112 is spaced apart from the plate 116 by a distance less than about 100 microns to form a fluid channel 120 between the membrane 112 and the plate 116. The housing 102 can then be attached or sealingly connected to the membrane 112. Embodiments of the present disclosure enable the cell culture dish 100 to be operable to receive a deposit of a cell culture and maintain a fluid and / or cells within the chamber 106. The cells within the chamber 106 may require a certain amount of oxygen or other gases (including air) to grow as desired by the user. The cell culture dish 100 can be operable to supply the required oxygen or other gases (including air) to the chamber 106. The pressurized gas can pass through the gas line 128, the one-way valve 130, the fluid channel 120 (between the membrane 112 and the plate 116), and flow out of the plurality of holes 122 into the chamber 106. When the gas passes through the fluid channel 120, the membrane 112 does not expand or flex in a manner such that the membrane 112 substantially expands into the chamber 106. In fact, the gas passing through the fluid channel 120 is limited to passing through the fluid channel 120 between the membrane 112 and the plate 116. In this regard, the membrane 112 does not occupy any significant volume within the chamber 106. In fact, all or substantially all of the volume of the chamber 106 can be used or occupied by the contents of the housing 102. The pressurized gas entering the chamber 106 exits from the plurality of holes 122 in the form of fine bubbles 132, and the fine bubbles rise through the liquid culture medium 133 contained in the housing 102 (e.g., the chamber 106). Although the membrane 112 does not have a conventional 3D conduit or channel designed to receive gas from the gas line 128, due to defects in the surfaces of the membrane 112 and the plate 116 that provide a low enough resistance to allow gas flow because the viscosity of the gas is extremely low, the gas can flow in the fluid channel 120 between the membrane 112 and the plate 116. In other words, embodiments of the membrane 112 and the plate 116 do not require the membrane 112 to deform or expand in response to pressure from the gas during use to create a fluid channel 120 that allows gas flow.

[0035] Embodiments of the membrane 112 have a surface roughness sufficient to allow gas or air to flow between the plate 116 and the document 112. In an embodiment, the surface roughness of the membrane 112 can be attributed to treating the surface of the membrane 112 with anti-clogging particles that protrude or extend from the surface of the membrane 112, thereby creating a rough surface that is not uniformly smooth. The roughness of the surface of the membrane 112 can be imparted, for example, by rollers on a membrane extrusion production line during manufacturing, or by polymer shrinkage when forming the holes 122 via a perforation method. Embodiments enable multiple holes 122 to be formed by mechanical hole punching or other methods such as, for example, laser ablation. Conversely, an embodiment can include that the plate 116 has a surface roughness obtained by treating the surface of the plate 116 during manufacturing such that the surface of the plate 116 does not have a uniformly smooth surface, but rather has multiple surface asperities that reduce surface-to-surface contact with the surface of the membrane 112. The roughness of one or both of the surfaces of the membrane 112 and the plate 116 provides a space between the membrane 112 and the plate 116 such that gas from the gas line 128 can pass through the fluid channel 120 between the membrane 112 and the plate 116 and through the holes 122. Embodiments include having one or more holes 122 based on a desired gas flow rate into the chamber 106. Embodiments include having an increased number of holes 122 to accommodate a higher gas or air flow rate into the chamber 106, and a smaller number of holes 122 to accommodate a lower gas or air flow rate desired by the user.

[0036] Embodiments of the bubbler 104 can be integrated into many types of vessels for cell culture, such as bioprocess cell expansion bags, spinner flasks, oxygenator vessels, bioreactors, and the like.

[0037] The present disclosure has been described in detail with particular reference to embodiments, but it should be understood that variations and modifications can be made within the spirit and scope of the present disclosure. Accordingly, the presently disclosed embodiments are to be considered in all respects only illustrative and not restrictive. The scope of the present disclosure is indicated by the appended claims, and all changes within the meaning and range of their equivalents are intended to be included therein.

Claims

1. A cell culture dish, comprising: A housing that defines a chamber operable to maintain a cell culture and an open end fluidly connected to the chamber, the open end being surrounded by a peripheral edge of the housing; An air bubbler coupled to the open end at the peripheral edge, the air bubbler including a membrane and a plate, the membrane including a first peripheral edge portion, the plate including a second peripheral edge portion, the first peripheral edge portion being sealingly connected to the second peripheral edge portion such that the membrane is spaced apart from the plate by less than about 100 micrometers to form a fluid channel between the membrane and the plate, the membrane further including a plurality of spaced-apart holes extending through the thickness of the membrane, the fluid channel being in fluid communication with the chamber through the plurality of holes.

2. The cell culture dish according to claim 1, wherein the thickness of the membrane is less than about 2.5 millimeters.

3. The cell culture dish according to claim 1 or claim 2, wherein the membrane comprises a polymer.

4. The cell culture dish according to any one of claims 1 to 3, wherein the diameter of each of the plurality of holes is less than about 1 millimeter.

5. The cell culture dish according to any one of claims 1 to 4, further comprising a gas line in fluid communication with the fluid channel.

6. The cell culture dish according to claim 5, further comprising a one-way valve operable to allow fluid to flow into the fluid channel in a first direction and prevent fluid from flowing out of the fluid channel in a second direction.

7. The cell culture dish according to claim 6, wherein the one-way valve is positioned in the gas line.

8. The cell culture dish according to claim 5, wherein the gas line is attached in a fixed manner between the membrane and the plate.

9. The cell culture dish according to any one of claims 1 to 8, wherein the membrane includes a first major surface facing the chamber, and the first major surface is non-planar.

10. The cell culture dish according to claim 9, wherein the first major surface is a treated surface configured to enhance and / or inhibit cell attachment to the first major surface.

11. The cell culture dish according to claim 10, wherein the treated first major surface includes a net negative surface charge.

12. The cell culture dish according to claim 10, wherein the treated first major surface includes a coating.

13. The cell culture dish according to any one of claims 1 to 12, wherein the wall of the housing is flexible.

14. The cell culture dish according to any one of claims 1 to 13, wherein the membrane and the plate are sealingly connected to each other by at least one of an airtight adhesive, heat sealing, hot melting, ultrasonic welding, or solvent bonding.

15. A method of bubbling a cell culture, comprising Depositing the cell culture into a cell culture dish, the cell culture dish including: A housing that defines a chamber operable to maintain the cell culture and an open end fluidly connected to the chamber, the open end being surrounded by a peripheral edge of the housing; A bubbler, which is connected to the open end at the peripheral edge, the bubbler includes a membrane and a plate, the membrane includes a first peripheral edge portion, the plate includes a second peripheral edge portion, and the second peripheral edge portion is sealingly connected to the first peripheral edge portion such that the membrane is spaced apart from the plate by less than about 100 μm to form a fluid channel between the membrane and the plate. The membrane further includes a plurality of spaced-apart holes extending through the thickness of the membrane, and the fluid channel is in fluid communication with the cavity through the plurality of holes; and allow gas to flow into the fluid channel, and the gas flows out of the plurality of holes into the housing.

16. The method according to claim 15, wherein the diameter of each of the plurality of holes is less than about 1 millimeter.

17. The method according to any one of claims 15 to 16, wherein the gas flows through a gas pipeline extending between the fluid channel and a gas source.

18. The method according to claim 17, wherein the gas flows through a one-way valve and then enters the fluid channel.

19. The method according to any one of claims 15 to 18, wherein the gas is a first gas, and the method further includes allowing a second gas different from the first gas to flow into the cavity.

20. The method according to any one of claims 15 to 19, wherein the membrane includes a first major surface facing the cavity, and the first major surface is treated to enhance and / or inhibit cell attachment to the first major surface.

21. The method according to any one of claims 15 to 20, wherein the deposition of the cell culture includes depositing an adherent cell culture and a suspension cell culture.

22. A method of forming a cell culture bubbler, which includes: positioning a membrane opposite to a plate including a second peripheral edge portion, the membrane includes a first peripheral edge portion and a plurality of holes extending through the thickness of the membrane; sealingly connecting the first peripheral edge portion to the second peripheral edge portion such that the membrane is spaced apart from the plate by a distance less than about 100 microns to form a fluid channel between the membrane and the plate.

23. The method according to claim 22, wherein the sealing includes at least one of an airtight adhesive, heat sealing, hot melting, ultrasonic welding or solvent bonding.

24. The method according to claim 22 or claim 23, wherein the thickness of the membrane is less than about 2.5 millimeters.

25. The method according to any one of claims 22 to 24, which further includes treating the surface of the membrane to enhance and / or inhibit cell attachment to the surface.

26. The method according to claim 25, wherein the treatment includes corona gas treatment.

27. The method according to claim 26, wherein the treated surface includes a net negative charge after the treatment.

28. The method according to claim 25, wherein the treatment includes applying a coating to the surface.

29. The method according to any one of claims 22 to 28, wherein the plurality of holes are formed by laser ablation.

30. The method according to claim 22, wherein the membrane comprises a first major surface facing the fluid channel and a second major surface opposite the first major surface, and the second major surface comprises one or more protrusions extending therefrom.

31. The method according to claim 22, wherein the membrane comprises a first major surface facing the fluid channel and a second major surface opposite the first major surface, and the second major surface is non-planar.

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