Laboratory apparatus and related methods

Abstract

The present disclosure relates to laboratory devices and systems, methods of use, and methods of manufacture. The laboratory apparatus of the present disclosure includes a chamber or container, and may also include one or more ports fluidly connected to the chamber. Thus, the laboratory equipment may be a closed system and thus may be particularly suitable for cell culture applications. In some embodiments, the laboratory device may include a gas permeable membrane. The chamber / container of the laboratory apparatus of the present disclosure can be conveniently filled with liquid and extracted from the chamber / container through a cover or adapter feature that allows the chamber to have a tilt or angle.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 405,040, filed on September 9, 2022, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to laboratory equipment, such as equipment for culturing, incubating or aggregating cells. More specifically, the present disclosure relates to laboratory equipment for culturing, incubating or aggregating cells on a large scale. Background Art

[0004] Two-dimensional (2D) culture of adherent cells in monolayers, such as using T-flasks, has long been the gold standard. Standard equipment has been developed that allows users to efficiently grow cells in dishes or well plates at a relatively low cost. In theory, cells grown in a 2D monolayer receive uniform amounts of nutrients and growth factors and can be easily lifted from their growth surface.

[0005] There are several limitations to 2D cell culture. For example, the formation of a monolayer results in reduced cell-cell interactions. In addition, the plastic surface used to support monolayer culture is much harder than the in vivo environment. While the use of hydrogels can mitigate some of the effects of cell culture on plastic, it does not completely eliminate the problem.

[0006] In contrast, three-dimensional (3D) culture may be a format that better recapitulates in vivo conditions in the in vitro culture of many cell types. Cells grown in a 3D environment experience enhanced cell-cell and cell-extracellular matrix interactions compared to 2D culture. Improved gene expression, cell junction formation, differentiation, and drug response may be other advantages of 3D culture for certain cell types.

[0007] However, there are several disadvantages in current 3D culture systems, such as poor reproducibility, high cost, and increased experimental complexity.

[0008] 3D culture takes different forms, including scaffold-based assemblies and cell-based assemblies. In scaffold-based assemblies, cells are combined with acellular substrates, such as embedded in hydrogels or porous biomaterials. In cell-based assemblies, cells may spontaneously assemble to form cell aggregates due to cell-to-cell affinity. 3D culture aggregates have been used for a wide range of applications, including expansion, modeling, drug screening, and tissue delivery.

[0009] Given the advantages of 3D culture systems, there is a need to develop cost-effective systems, devices, and methods that can reproducibly achieve these advantages while overcoming the various shortcomings of current systems and devices. Summary of the invention

[0010] One aspect of the present disclosure provides a laboratory device. The laboratory device of this aspect may include: a housing having one or more side walls extending substantially orthogonally from a planar member; and a gas-permeable membrane sealingly engaged with the housing, the gas-permeable membrane and the housing forming a container, the container having a chamber defined by a top wall and a bottom wall, the top wall and the bottom wall being connected and bounded by one or more side walls.

[0011] The laboratory device of the present disclosure may also include a first port and an opposite second port, each port being in fluid communication with the chamber. In one embodiment, the first port and the second port are opposite along a diagonal or diameter direction. In one embodiment, the first port and the second port extend through the top wall.

[0012] In one embodiment, the laboratory device is closed and / or sealed.

[0013] In one embodiment, the diameter of the second port is the same as or greater than the diameter of the first port. In one embodiment, the diameter of the first port is between about 3 mm and 5 mm. In one embodiment, the diameter of the second port is between about 3 mm and 12 mm. In one embodiment, the diameter of the first port is different from the diameter of the second port.

[0014] The laboratory device of the present disclosure may also include a plurality of micropatterned features in the bottom wall of the chamber. In one embodiment, the micropatterned features are cylindrical, inverted cones, inverted truncated cones, inverted pyramids, or inverted truncated pyramids. In one embodiment, the depth of each micropatterned feature is between about 100 μm and 4 mm. In one embodiment, the width or diameter of each micropatterned feature taken in a plane across its opening is between about 100 μm and 5 mm. In one embodiment, the aspect ratio of each micropatterned feature is less than 1.

[0015] In one embodiment, the gas permeable membrane forms the bottom wall and the plurality of micropatterned features are formed in or on the gas permeable membrane.

[0016] In one embodiment, the gas permeable membrane forms the top wall, the planar member forms the bottom wall, and a plurality of micropatterned features are formed in or on the planar member.

[0017] The laboratory device of the present disclosure may also include a frame located outside the chamber and overlapping at least the periphery of the gas permeable membrane. In one embodiment, the frame includes at least one bracket against the gas permeable membrane to limit the stretching of the gas permeable membrane and the increase of the chamber volume when the chamber is filled with fluid.

[0018] In one embodiment, the first port and the second port are formed in and / or across opposing corners or edges of the frame.

[0019] In one embodiment, the first port and the second port are defined by mating frame wall portions and connecting wall portions, respectively, to form a first port reservoir and a second port reservoir. In one embodiment, the height of the connecting wall portion is lower than the height of the frame wall portion.

[0020] The laboratory equipment of the present disclosure may also include a lid having a continuous skirt extending orthogonally downward from its upper plane. In one embodiment, the height of the skirt is the smallest at a first edge or corner of the lid, and the height of the skirt is the largest at an opposite second edge or corner of the lid.

[0021] In one embodiment, the first edge or corner of the cover, the second edge or corner of the cover, the first port, and the second port are positioned along a common axis when viewed from above and when the cover is in position covering the housing.

[0022] In one embodiment, the bottom wall of the container is inclined when the housing is positioned on the lid and when the skirt rests on a horizontal surface. In one embodiment, the bottom wall is inclined about an inclined axis that is orthogonal to the common axis. In one embodiment, the bottom wall is inclined between 0 and 45 degrees, and preferably less than 10 degrees, and more preferably 5 degrees or less.

[0023] In one embodiment, at least the frame and the housing are made of polymers independently selected from polystyrene (PS), polymethylpentene (PMP), polycarbonate (PC), polymethyl methacrylate (PMMA), silicon, silicone-based or styrene block copolymers. In one embodiment, the breathable membrane is made of polystyrene (PS), polymethylpentene (PMP), polycarbonate (PC), polymethyl methacrylate (PMMA), silicon, silicone-based or styrene block copolymers.

[0024] Another aspect of the present disclosure provides a laboratory device. The laboratory device of this aspect may include a container, the container having: one or more side walls extending upwardly and substantially orthogonally from a bottom wall; one or more restrictions surrounding the one or more side walls, the one or more restrictions extending from the bottom wall by a non-constant and shorter distance relative to the one or more side walls; and a lid having a continuous skirt extending downwardly and orthogonally from an upper plane thereof, the height of the skirt being the smallest at a first edge or corner of the lid, and the height of the skirt being the largest at an opposite second edge or corner of the lid.

[0025] In one embodiment, the bottom wall lies in a substantially horizontal plane when the container rests on a horizontal surface, and the upper plane of the lid lies in a plane parallel to the bottom wall when the skirt rests on the one or more restraints, and the bottom wall is inclined relative to the horizontal surface when the underside of the container lies on the upper plane of the lid when the skirt rests on the horizontal surface. In one embodiment, the bottom wall is inclined about an inclination axis that is orthogonal to an axis passing through a first edge or corner and an opposite second edge or corner of the lid (when viewed from above). In one embodiment, the bottom wall is inclined between 0 and 45 degrees, and preferably less than 10 degrees, and more preferably 5 degrees or less.

[0026] The laboratory apparatus of the present disclosure may further include a gas permeable membrane sealingly secured to one or more of the side walls.

[0027] In one embodiment, the gas permeable membrane forms the bottom wall.

[0028] In one embodiment, the breathable membrane is spaced apart from the bottom wall and lies in a plane parallel to the plane of the bottom wall (eg, the membrane forms the top wall).

[0029] The laboratory device of the present disclosure may also include a plurality of micropatterned features in the bottom wall of the container. In one embodiment, the micropatterned features are cylindrical, inverted cones, inverted truncated cones, inverted pyramids, or inverted truncated pyramids. In one embodiment, the depth of each micropatterned feature is between about 100 μm and 4 mm. In one embodiment, the width or diameter of each micropatterned feature taken in a plane across its opening is between about 100 μm and 5 mm. In one embodiment, the aspect ratio of each micropatterned feature is less than 1.

[0030] The laboratory device of the present disclosure may also include a first port and an opposite second port, each port being in fluid communication with a chamber formed between the bottom wall and the gas permeable membrane and bounded by one or more side walls. In one embodiment, the diameter of the second port is the same as or greater than the diameter of the first port.

[0031] The laboratory device of the present disclosure may also include a frame located outside the chamber and overlapping at least the periphery of the gas permeable membrane. In one embodiment, the frame includes at least one bracket against the gas permeable membrane to limit the stretching of the gas permeable membrane and the increase of the chamber volume when the chamber is filled with fluid.

[0032] In one embodiment, the first port and the second port traverse and / or are disposed in opposite corners or edges of the frame. In one embodiment, the first port and the second port are defined by mating frame wall portions and connecting wall portions, respectively, forming a first port reservoir and a second port reservoir. In one embodiment, the height of the connecting wall portion is lower than the height of the frame wall portion.

[0033] Another aspect of the present disclosure provides a method for using the device of the present disclosure in a laboratory assay, experiment or incubation. For example, an assay, experiment or incubation may involve cells or other types of analytes, such as biomolecules. In an embodiment involving cells, the method may involve culturing or incubating cells, such as forming non-adherent cell aggregates. Regardless of the process used for the device of the present disclosure, liquid can be conveniently added and / or removed from its container / chamber by tilting the device (such as in conjunction with a provided lid). In certain embodiments, the method of the present disclosure relates to a closed and / or sealed device, particularly when the method relates to cells. In such embodiments, a liquid (e.g., a cell suspension and / or culture medium) can be introduced into a closed / sealed chamber via a port. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] To better understand the various embodiments described herein, and to more clearly show how these various embodiments may be implemented, reference will be made, by way of example, to the accompanying drawings which illustrate at least one example embodiment and which will now be described. The drawings are not intended to limit the scope of the teachings described herein.

[0035] Figure 1 Various views of an exemplary device of the present disclosure are shown. A perspective side view (A), a cross-sectional view (B), and a top view (C) are depicted.

[0036] Figure 2 A perspective side view (A) and a cross-sectional view (B) taken along plane "A" of the base / housing of one embodiment of the device of the present disclosure are shown.

[0037] Figure 3 Exploded views (A) and enlarged exploded views (B) of various embodiments of the apparatus of the present disclosure are shown, while also emphasizing potential methods of manufacture.

[0038] Figure 4 A perspective view (A) and a cross-sectional view (B) of different embodiments of the apparatus of the present disclosure are shown.

[0039] Figure 5 Cross-sectional views of various embodiments of micropatterned features are shown.

[0040] Figure 6 Various embodiments of frames and supports included in the devices of the present disclosure are shown (A) and their corresponding impact on the volume of the device chamber (B).

[0041] Figure 7 Different embodiments of ports are shown in panels (A) and (B).

[0042] Figure 8Images showing fluid extraction operation of the device of the present disclosure at a tilt of 0° (A), 1° (B), 2° (C), or 3° (D).

[0043] Fig. 9 The relationship of the base / housing and lid of an exemplary device is shown. A partially exploded view is shown in (A), with the base / housing suspended above the lid. A perspective view (B), a front view (C), and a side view (D) of the base / housing resting on the lid at an angle.

[0044] Fig.10 The relationship of the base / housing and lid of an exemplary device is shown. A partially exploded view is shown in (A) with the lid suspended above the base / housing. A perspective view (B) and a front view (C) of the lid resting on the base / housing.

[0045] Fig.11 A method of making a breathable film having a plurality of thermoformed micropatterned features therein is shown in accordance with the present disclosure. DETAILED DESCRIPTION

[0046] The present disclosure relates to laboratory equipment (e.g., cell culture equipment), systems, and methods related to their use or manufacture. The equipment of the present disclosure can be used to culture, incubate, and / or aggregate cells. In some embodiments, the laboratory equipment includes a micropatterned surface (e.g., a surface with multiple micropores). In one embodiment, the laboratory equipment includes a closed or sealed chamber. In one embodiment, multiple separate equipment can be used to achieve scale-out beyond the limitations of a single laboratory equipment (e.g., cell culture equipment).

[0047] As used in the present disclosure, the term "laboratory equipment" refers to equipment used in a laboratory that can perform experiments or measurements, such as experiments or measurements performed on liquids that may contain analytes, biomolecules or cells. In one embodiment, the laboratory equipment is a cell culture device. Therefore, as used in the present disclosure, the term "cell culture device" refers to a device that can inoculate and incubate cells. The cells inoculated into the cell culture device of the present disclosure are not particularly limited, and can be adherent cells or non-adherent cells. The cells placed in the chamber of the disclosed device can be primary cells, cell lines, cancer cells, pluripotent stem cells, or cells differentiated from pluripotent stem cells, etc. In one embodiment, the chamber of the laboratory equipment, especially at least its surface perpendicular to gravity, is not suitable for 2D culture of monolayer adherent cells. In such embodiments, cells that are usually adherent to the chamber may form suspended cell aggregates, embryoid bodies or organoids. In one embodiment, at least one surface (perpendicular to gravity) of the internal chamber or container is modified to include multiple micro-patterned features (e.g., micropores), as further described below.

[0048] As used in this disclosure, the terms "cell aggregate" or "aggregate" refer to a group of cells that coalesce to form an interconnected mass. Cells may spontaneously form aggregates, or may be forced to form aggregates. When multiple cells are forced into direct contact, they may be forced to coalesce into aggregates. Aggregate formation can be influenced by positioning multiple cells on a surface topology. In embodiments where the cells are adherent cells, it is important that their tendency to self-aggregate overcomes their tendency to attach to a non-cellular surface, such as a cell culture surface.

[0049] Unless otherwise defined, scientific and technical terms used in the devices, systems and methods described herein shall have the meanings commonly understood by those of ordinary skill in the art. The terms used herein are only used to describe specific embodiments and are not intended to limit the scope of the present invention, which is defined only by the claims.

[0050] Equipment and systems

[0051] One aspect of the present disclosure provides laboratory equipment, such as cell culture equipment. In one embodiment, the equipment of the present disclosure is a closed system or a sealed system. In other words, the internal chamber of the equipment is not directly exposed to the external environment, but is sealed relative to the external environment. However, the closed cell culture equipment may include a gas exchange device to introduce oxygen into its internal chamber. In addition, considering the demand for nutrients and / or growth factors for cultured cells, the closed cell culture equipment will include a device for introducing nutrients and / or growth factors (preferably contained in the cell culture medium) into its internal chamber.

[0052] refer to Figure 1 , the apparatus 1 of the present disclosure may include a housing 3 that defines or cooperates to define a container and / or chamber 5. The housing 3 may include one or more side walls 7 that extend substantially orthogonally from a substantially planar member 9. In one embodiment, the one or more side walls 7 and the planar member 9 are integral. In one embodiment, the one or more side walls 7 and the planar member 9 are at least a two-piece structure.

[0053] refer to Figure 1 B and Figure 2 , the housing 3 may include a first shoulder 12. The first shoulder 12 extends orthogonally or substantially orthogonally away from the one or more side walls 7. More specifically, the shoulder 12 may extend from a point intermediate the bottom and apex of the one or more side walls 7 toward the interior of the chamber / container 5. In one embodiment, the shoulder 12 is formed on or in the inner surface of the one or more side walls 7 (e.g., the surface of the one or more side walls on the chamber / container side). In one embodiment, the shoulder 12 forms a perimeter within the chamber / container 5.

[0054] The shoulder 12 may be of any width. w . For example, the shoulder 12 provides sufficient surface area for adhesive to be applied thereto, but is not so wide as to substantially reduce the volume of the chamber / container 5. In one embodiment, the width of the shoulder 12 is about 1 mm. In one embodiment, the width of the shoulder 12 is about 2 mm. In one embodiment, the width of the shoulder 12 is about 3 mm. In one embodiment, the width of the shoulder 12 is about 4 mm. In one embodiment, the width of the shoulder 12 is about 5 mm. In a preferred embodiment, the width of the shoulder 12 is between about 1 mm and 5 mm.

[0055] The shoulder 12 may be of any height. h ; but should be sufficient to maintain the desired volume within the container / chamber 5. In one embodiment, the height of the shoulder 12 is about 2 mm. The height of the shoulder 12 (measured from the bottom wall) can be about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 12 mm, about 14 mm, about 16 mm, about 18 mm, or about 20 mm. In a preferred embodiment, the height of the shoulder 12 (measured from the bottom wall) is between about 5 mm and 20 mm.

[0056] The housing 3 can be made of any material, but preferably comprises a polymer. In one embodiment, the housing 3 is made of a material suitable for molding techniques (such as injection molding). Non-limiting examples of the manufacturing materials of the housing 3 include: polystyrene (PS), polymethylpentene (PMP), polycarbonate (PC), polymethyl methacrylate (PMMA), silicon, silicone-based materials or copolymers, such as styrene block copolymers.

[0057] The device 1 may also include a gas permeable membrane 15. The gas permeable membrane 15 cooperates with the housing 3 to form a container / chamber 5, which may be defined by a top wall and a bottom wall connected and bounded by at least a portion of one or more side walls 7. The gas permeable membrane 15 may be made of any material as long as oxygen and other gases can easily diffuse through the membrane (into the container / chamber 5) and the material is non-toxic or will not damage biological molecules or cells, or will not emit gaseous toxins or pollutants into the container / chamber 5.

[0058] The breathable membrane 15 can be made of any material, but preferably comprises a polymer. In one embodiment, the breathable membrane 15 is made of a material suitable for extrusion or molding. Non-limiting examples of materials for making the breathable membrane 15 include: PS, PMP, PC, SBS / SEBS, silicon, silicone-based materials or copolymers, such as styrene block copolymers.

[0059] In one embodiment, the air permeable membrane 15 forms the top wall of the chamber 5 (eg Figure 1 and Figure 3As shown), and in this case the bottom wall of the chamber 5 can be a planar member 9. Thus, the breathable membrane 15 can be bonded or otherwise attached to one or more side walls 7, or more specifically to the shoulder 12. The breathable membrane can be attached to the housing 3 by other means, such as by any means known to those skilled in the art. In one embodiment, the breathable membrane 15 is attached to the housing 3 (e.g., one or more side walls 7 or shoulder 12) to ensure a sealed joint (e.g., leakproof) under normal use conditions (e.g., incubated at 37° to 75°). In addition, the choice of the adhesive may be important from the perspective of biocompatibility and / or the ability to attach / bond different materials.

[0060] exist Figure 3 In the exemplary embodiment shown in FIG. 1A , an adhesive 16 a is applied to the shoulder 12 to fix the breathable membrane 15 to the housing 3 . The adhesive can be any type of adhesive as long as it can bond the materials of the shoulder 12 and the breathable membrane 15 . In one embodiment, the adhesive is a double-sided tape. In one embodiment, the adhesive is glue.

[0061] The device 1 may also include a frame 17. The frame 17 may provide one or more structural attributes and / or one or more functional attributes. Potential roles of the frame 17 may include facilitating the securing of the gas permeable membrane 15 to the housing 3 (such as to the shoulder 12); stabilizing the gas permeable membrane 15 to prevent it from stretching when the chamber 5 is filled with liquid, and supporting / bonding holes or ports through which liquid can be introduced into or withdrawn from the chamber 5. Thus, in one embodiment, the frame 17 may cooperate with the housing 3 (such as the shoulder 12) and the adhesives 16a and 16b to attach or secure the gas permeable membrane 15.

[0062] exist Figure 3 In the exemplary embodiment shown in FIG. 1B , the breathable membrane 15 is fixed to the housing 3 by welding (such as by ultrasonic welding). The fixing of the breathable membrane 15 to the housing 3 by ultrasonic welding can be facilitated by a frame 17 (described further below) outside the chamber 5, which is placed around at least the periphery of the breathable membrane 15 or overlaps / is located above it, so that the membrane 15 is sandwiched between the shoulder 12 and the frame 17. In such embodiments, the frame 17 and the shoulder 12 may include mating ribs 19 that contact each other to further facilitate ultrasonic welding. In other embodiments, the frame 17 and the shoulder 12 may include matable ribs and grooves, respectively, which cooperate to fix the breathable membrane 15 to the housing 3.

[0063] In one embodiment, the gas permeable membrane 15 forms the bottom wall of the chamber 5 (eg Figure 4), and in this case the top wall can be a planar member 9. Thus, the breathable membrane 15 can be bonded or otherwise attached to one or more side walls 7. In one embodiment, the breathable membrane 15 can be directly bonded or otherwise attached to the edge of one or more side walls 7. In one embodiment, the shoulder and / or frame features substantially as described above (except inverted) can mediate the attachment of the breathable membrane 15 to the housing 3. In one embodiment, the breathable membrane 15 is attached to the housing 3 in a specific manner to ensure a sealed joint (e.g., leakproof) under normal use conditions, and the attachment method can be as described above or in any other manner known to those skilled in the art. In addition, the choice of adhesive may be important from the perspective of biocompatibility and / or the ability to attach / bond different materials.

[0064] The breathable membrane 15 is not particularly limited in size, especially its thickness, as long as the gas can diffuse through the membrane to the same or better extent than the material of the microporous plate or cell culture bottle. In one embodiment, the thickness of the breathable membrane 7 is between about 0.05mm and 1mm. In one embodiment, the thickness of the breathable membrane 7 is between about 0.1mm and 0.8mm. In one embodiment, the thickness of the breathable membrane 7 is between about 0.15mm and 0.7mm. In one embodiment, the thickness of the breathable membrane 7 is between about 0.2mm and 0.65mm. In one embodiment, the thickness of the breathable membrane 7 is between about 0.25mm and 0.6mm. In one embodiment, the thickness of the breathable membrane 7 is about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm or thicker.

[0065] In one embodiment, the elements defining or cooperating to define the chamber 5 may provide different permeabilities. In one embodiment, only the gas permeable membrane 15 is permeable to gas or permeable to a sufficient amount of gas on the time scale of (cell) culture, incubation or aggregation. More specifically, the housing 3 may not be permeable to gas or may only be permeable to an insufficient amount / volume of gas on the time scale of (cell) culture, incubation or aggregation.

[0066] The device 1 may also include at least one port, preferably more than one port. In one embodiment, the device 1 includes a first port 20 and a second port 25 ( Figure 1 and Figure 4 ). The first port 20 and the second port 25 are each in fluid communication with the chamber 5, but can still be inserted or insertable to prevent the contents from escaping from the chamber 5 and / or to protect the contents of the chamber 5 from the environment outside the device 1.

[0067] The positioning of the first port 20 and / or the second port 25 may depend on how the chamber 5 is configured relative to the positioning of the gas permeable membrane 15. For example, when the gas permeable membrane 15 forms the bottom wall, the first port 20 and / or the second port 25 may pass through the top wall of the chamber 5. As an additional example, when the membrane forms the top wall of the chamber 5, the first port 20 and / or the second port 25 may pass through the gas permeable membrane 15, or a corresponding orifice therein. In one embodiment, the first port 20 and / or the second port 25 may pass through one or more side walls 7.

[0068] In one embodiment, the first port 20 and / or the second port 25 are located on, through, or extend through a top wall (e.g., membrane 15 or planar member 9, depending on the configuration) of the device 1. Thus, the first port 20 and / or the second port 25 mate with a corresponding hole through the top wall. In one embodiment, one or both ports may extend about 0.5 to 2 mm toward the bottom wall. In one embodiment, the first port 20 and / or the second port 25 may be located on, extend through, or through one or more side walls 7 of the housing 3.

[0069] The diameter of the first port 20 (and in some embodiments, the diameter of the hole that cooperates with the port) does not hinder the passage of air therethrough, but otherwise the diameter is not particularly limited. In one embodiment, the diameter of the first port 20 (and in some embodiments, the diameter of the hole that cooperates with the port) is not less than 3 mm. In one embodiment, the diameter of the first port 20 (and in some embodiments, the diameter of the hole that cooperates with the port) is between about 3 mm and 5 mm. In one embodiment, the diameter of the first port 20 (and in some embodiments, the diameter of the hole that cooperates with the port) is about 4 mm.

[0070] The diameter of the second port 25 (and in some embodiments, the diameter of the hole that cooperates with the port) does not hinder the passage of liquid (such as cell culture medium). In one embodiment, the diameter of the second port 25 (and in some embodiments, the diameter of the hole that cooperates with the port) is the same as or greater than the diameter of the first port 20. In one embodiment, the diameter of the second port 25 (and in some embodiments, the diameter of the hole that cooperates with the port) is between about 3 mm and 12 mm. In one embodiment, the diameter of the second port 25 (and in some embodiments, the diameter of the hole that cooperates with the port) is about 10 mm or about 12 mm. When a serological pipette or an object of similar size is used to introduce or withdraw liquid from the chamber 5, it may be preferable to use a relatively large diameter (e.g., 10 mm or 12 mm). When a pipette smaller than a serological pipette is used, or when a tube connected to a pump is used to introduce or withdraw liquid from the chamber 5, it may be preferable to use a relatively small diameter (e.g., between 3 mm and 6 mm).

[0071] The port included in the device 1 can be made of any material. Typically, the port is made of a polymer, which may be thermoformed. In one embodiment, the port can be or include a Luer connector.

[0072] In a preferred embodiment, the device 1 includes two ports (e.g., a first port 20 and a second port 25). In such embodiments, the ports may be located in or near opposite corners or edges of the device 1 (e.g., Fig. 9 and Fig.10 More specifically, the ports may be located in or near diagonally or diametrically opposed corners or edges of the device 1 (such as the top wall).

[0073] In one embodiment, the device 1 further comprises a plurality of micro-patterned features 30 (see Figure 4 and Figure 5 ). The size of the plurality of micropatterned features 30 can be designed to accommodate a plurality of cells. Therefore, in some embodiments, the plurality of micropatterned features 30 are formed or disposed on an inner surface of the chamber 5 perpendicular to the direction of gravity, such as a bottom wall (e.g., a planar member or a gas-permeable membrane, depending on the configuration). In one embodiment, the cells accommodated in the corresponding micropatterned features will aggregate into (non-adherent) cell aggregates. In one embodiment, the bottom wall of the chamber 5 formed with the plurality of micropatterned features itself does not support the anchorage-dependent growth of cells.

[0074] In the embodiment where the gas permeable membrane 15 forms the bottom wall of the chamber 5, a plurality of micro-patterned features 30 may be formed in or on the gas permeable membrane 15. The micro-patterned gas permeable membrane may be manufactured using a thermoforming process, such as embossing (see, for example, Fig.11 In such embodiments, the plurality of micropatterned features 30 may descend from the upper plane of the gas permeable membrane 15 .

[0075] In embodiments where the gas permeable membrane 15 forms the top wall of the chamber 5, a plurality of micro-patterned features 30 may be formed in or on the bottom wall (e.g., the planar member 9). The micro-patterned bottom wall may be manufactured using a thermoforming process such as embossing or liquid molding, or by stamping or etching. In such embodiments, the plurality of micro-patterned features 30 may descend from the upper plane of the bottom wall (e.g., the base of the housing 3).

[0076] Each micropatterned feature may have the same shape. In one embodiment, features of different shapes may be included in the plurality of micropatterned features 30. Regardless, the plurality of micropatterned features 30 may be cylindrical, inverted cone, inverted truncated cone, inverted pyramid, or inverted truncated pyramid. In a preferred embodiment, the plurality of micropatterned features 30 are inverted pyramids or inverted truncated pyramids ( Figure 5 ).

[0077] The plurality of micropatterned features 30 may be arranged in any manner; however, when seeking to maximize the density of micropatterned features on a surface (surface for a given surface area), a more efficient arrangement may be desired. In one embodiment, the plurality of micropatterned features 30 are arranged in rows and columns. In one embodiment, the plurality of micropatterned features 30 are arranged in continuous rows and columns (e.g., in a grid pattern when viewed from the top).

[0078] In one embodiment, the spacing between adjacent individual features (e.g., micropores) is minimized. When one wants to maximize the scale of a culture, experiment, or assay, a relatively large space (i.e., non-minimum space) between adjacent individual features (e.g., micropores) can lead to inefficiencies. In one embodiment, the spacing between adjacent micropatterned features in a plurality of micropatterned features 30 is minimized. In the context of a cell culture device, when the spacing between adjacent individual features is equal to or greater than the cell diameter, some cells in the chamber 5 may not be deposited in the feature, but instead stay on the spacing. In one embodiment, the ridges between adjacent individual features (e.g., micropores) are smaller than the diameter of the cell (e.g., less than 15 μm, less than 10 μm, less than 5 μm, less than 3 μm, less than 2 μm, or < less than 1 μm). In one embodiment, adjacent micropatterned features in a plurality of micropatterned features are spaced apart at equal intervals.

[0079] In the context of use of the apparatus 1 for cell culture, it may be desirable to restrict at least the length and width of the apparatus 1 to the ANSI plate format.

[0080] The size of each of the plurality of micropatterned features 30 is not particularly limited. In one embodiment, each of the plurality of micropatterned features 30 is sized to accommodate more than one cell. In some embodiments, each of the plurality of micropatterned features 30 is sized to accommodate up to 100 cells. In one embodiment, each of the plurality of micropatterned features 30 is sized to accommodate up to 1000 cells. In one embodiment, each of the plurality of micropatterned features 30 is sized to accommodate up to 5000 cells. In one embodiment, each of the plurality of micropatterned features 30 is sized to accommodate up to 10000 cells. In one embodiment, each of the plurality of micropatterned features 30 is sized to accommodate more than 10000 cells.

[0081] In one embodiment, the depth MP of each micropatterned feature is dBetween about 50 μm and 4 mm. In one embodiment, the depth of each micropatterned feature is between about 75 μm and 3 mm. In one embodiment, the depth of each micropatterned feature is between about 100 μm and 2 mm.

[0082] In one embodiment, the width MP of each micropatterned feature taken in a plane across its opening is w In one embodiment, each micropatterned feature has a width or diameter in a plane across its opening of between about 50 μm and 5 mm. In one embodiment, each micropatterned feature has a width or diameter in a plane across its opening of between about 75 μm and 3 mm. In one embodiment, each micropatterned feature has a width or diameter in a plane across its opening of between about 100 μm and 2 mm.

[0083] In one embodiment, such as when the micropatterned features are inverted pyramids / cones or inverted truncated pyramids / cones, each micropatterned feature may have a width of about 200 μm taken in a plane across its opening, and the depth of such micropatterned features may be between about 100 and 150 μm. In a specific embodiment, the depth of such micropatterned features may be about 140 μm.

[0084] In one embodiment, such as when the micropatterned features are inverted pyramids / cones or inverted truncated pyramids / cones, each micropatterned feature may have a width of about 400 μm taken in a plane across its opening, and the depth of such micropatterned features may be between about 250 and 300 μm. In a specific embodiment, the depth of such micropatterned features may be about 280 μm.

[0085] In one embodiment, such as when the micropatterned features are inverted pyramids / cones or inverted truncated pyramids / cones, each micropatterned feature may have a width of about 800 μm taken in a plane across its opening, and the depth of such micropatterned features may be between about 350 and 400 μm. In a specific embodiment, the depth of such micropatterned features may be about 390 μm.

[0086] MP d and MP w The relationship can be any ratio of the corresponding dimensions provided herein.

[0087] Notwithstanding the above, the aspect ratio (i.e., the ratio between the depth and width of the opening across the micropatterned features) is not particularly limited. In some embodiments, it may be desirable to minimize the destruction of cells or aggregates within the micropatterned features, and thus aspect ratios greater than 1, greater than 2, greater than 3, or greater than 4 may be preferred. However, the higher the aspect ratio, the more difficult it may be to retrieve the contents of each micropatterned feature. In some embodiments, it may be desirable to preferentially recover cells or aggregates from within the micropatterned features, and thus aspect ratios of about 1 or less may be preferred.

[0088] In one embodiment, the aspect ratio of each micropatterned feature in the plurality of micropatterned features 30 is 1 or less than 1. In one embodiment, the aspect ratio of each micropatterned feature in the plurality of micropatterned features 30 is between 0.5 and 1.

[0089] In embodiments of the device 1 comprising a plurality of micropatterned features (e.g. microwells), the number of individual such features is not particularly limited. In fact, the number of individual features is limited by their size and the size of the device 1 (particularly its bottom wall). In embodiments where the device 1 occupies an area equivalent to or substantially equivalent to a typical microwell plate (ANSI 1-2004, 127.76 x 85.48 mm), more than 100,000 individual features may be provided, depending on their size. For example, where the width of each pyramid (or truncated pyramid) feature (across its opening) is 200 μm, approximately 125,000 individual features may be provided in a single device 1, and where the width of each pyramid (or truncated pyramid) feature (across its opening) is 400 μm, approximately 35,000 individual features may be provided in a single device 1.

[0090] refer to Figure 1 , Figure 3 , Figure 6 and Figure 7 , the device 1 may further include a frame 17 outside the chamber 5, the frame overlapping / located on at least the periphery of the gas porous membrane 15. As described above, the frame 17 may facilitate attachment of the gas permeable membrane 15 to the housing 3. The frame 17 may also provide support for the gas porous membrane 15, such as by limiting stretching of the gas porous membrane and a corresponding increase in the volume of the chamber (when or because the chamber is filled with fluid).

[0091] In one embodiment, the frame 17 includes at least one support 40. In one embodiment, the at least one support 40 spans opposite or adjacent edges of the frame 17 and is located above (or below, depending on the configuration of the device 1). In one embodiment, the device 1 includes a second support 42. In one embodiment, the second support 42 spans the same or different opposite or adjacent edges of the frame 17 and is located above (or below, depending on the configuration of the device 1) the gas permeable membrane 15. In one embodiment, the second support 42 intersects with the first support 40. In one embodiment, the second support 42 does not intersect with the first support 40. In embodiments where the device 1 includes at least one support (and an optional second support), the support can support the gas permeable membrane 7 and protect its integrity. For example, when the gas permeable membrane 7 forms the top wall of the chamber 5, it may bubble (expand upward) due to the force of the liquid filling the chamber 5, thereby creating a local liquid (e.g., cell culture medium) height difference within the chamber 5 and may create uneven waste, oxygen, nutrient and / or growth factor gradients. In fact, Figure 7 It shows that as the number of brackets decreases, the liquid volume in chamber 5 increases.

[0092] The frame 17 can be made of any material, but preferably comprises a polymer. In one embodiment, the frame 17 is made of a material suitable for molding techniques (such as injection molding). Non-limiting examples of materials for making the frame 17 include: PS, PMP, PC, PMMA, SBS / SEBS, silicon, silicone-based materials or copolymers, such as styrene block copolymers.

[0093] In one embodiment, the membrane 15, the frame 17 and the housing 3 are made of the same material. In one embodiment, the membrane 15 is made of a different material than the frame 17 and the housing 3. In one embodiment, the membrane 15, the frame 17 and the housing 3 are all made of different materials.

[0094] In one embodiment, the frame 17 and / or the brackets 40, 42 are attached to the breathable membrane 15 using an adhesive (e.g., adhesive 16b). The adhesive can be any type of adhesive as long as it can bond the materials of the frame 17 (and / or the bracket) and the breathable membrane 15 together. In one embodiment, the adhesive is a double-sided tape. In one embodiment, the adhesive is glue. In one embodiment, the adhesive used to attach the frame 17 to the breathable membrane 15 is the same as the adhesive used to attach the shoulder 12 to the breathable membrane 15.

[0095] The size or thickness of the at least one first bracket 40 (and the second bracket 42, if present) against the gas permeable membrane 15 may affect the diffusion of oxygen thereon, or the distribution of diffused oxygen throughout the chamber 5. Generally, when the thickness of the surface of the at least one first bracket 40 (and the second bracket 42, if present) against the gas permeable membrane 15 is in the millimeter range (e.g., between 1 and 10 mm), a more uniform oxygen distribution can be observed.

[0096] In certain embodiments of the device 1, the first port 20 and the second port 25 can be configured in or integral with the frame 17 so as to traverse the frame 17. In one embodiment, the first port 20 and the second port 25 are located in (and traverse) diagonally opposite corners or edges of the frame 17 ( Figure 1 and Figure 7 In one embodiment, the first port 20 and the second port 25 are integrally located in (and across) diagonally opposite corners or edges of the frame 17 ( Figure 1 and Figure 7 In one embodiment, the first port 20 and the second port 25 are attached to holes molded into diagonally opposite corners or edges of the frame 17 ( Figure 1 and Figure 7 ).

[0097] The frame 17 can include a peripheral frame wall 45. In one embodiment, the frame wall 45 has the same or substantially the same width as the shoulder 12. In one embodiment, the frame wall 45 has a smaller width than the shoulder 12. In embodiments where the frame wall 45 has a smaller width than the shoulder 12, the lack of width can be compensated by a flange connected to the frame wall 45 and extending orthogonally to overlap at least a portion or all of the width of the shoulder 12.

[0098] When positioned on the shoulder 12, the frame wall 45 extends from the shoulder 12 toward or to the apex of the one or more side walls 7. In one embodiment, when the frame 17 is positioned on the shoulder 12, the height of the frame wall 45 extends to the apex of the one or more side walls 7. In one embodiment, when the frame 17 is positioned on the shoulder 12, the height of the frame wall 45 does not extend to the apex of the one or more side walls 7.

[0099] In embodiments where the first and second ports 20, 25 are disposed in or on the frame 17, they may be positioned adjacent to opposing edges or corners of the frame 17 ( Figure 7). Thus, the first port 20 and the second port 25 may be at least partially defined by a portion of the frame wall 45, and in this case, the first port 20 and the second portion 25 may be completely surrounded by a connecting wall 47 that cooperates with the portion of the frame wall 45, respectively. Thus, the first port 20 and the second port 25 may be surrounded by the frame wall portion 45 and the connecting wall 47, respectively, to form a port reservoir 49. The port reservoir 49 may help limit and / or direct fluid to be introduced into or removed from the chamber 5.

[0100] In one embodiment, the height of the connecting wall portion 47 is the same or substantially the same as the height of the frame wall 45. That is, the apex of the connecting wall portion 47 is the same or substantially the same as the apex of the frame wall 45. In one embodiment, the height of the connecting wall portion 47 is lower than or shorter than the height of the frame wall portion 45 ( Figure 7 ). A height of the connecting wall portion 47 that is lower than or shorter than the height of the frame wall portion 45 can advantageously reduce the siphoning of liquid out of the device 1 from the port reservoir 49 or from the chamber 5 via the port reservoir 49.

[0101] In use, liquid (e.g., culture medium containing a cell suspension) may be introduced into the chamber 5 via the second port 25 (e.g., a liquid port). To avoid or limit the formation of bubbles, the first port 20 (e.g., an exhaust port) allows air to be drawn out of the chamber 5 as it is displaced by the liquid. Figure 8 ). Therefore, the minimum diameter of the first port 20 as described above can be selected so that the liquid does not form a seal on the hole by surface tension.

[0102] The inventors have also unexpectedly discovered numerous advantages of the disclosed apparatus 1. For example, in a closed (e.g., sealed) configuration of the apparatus 1, the chamber 5 can be filled with a liquid (e.g., a culture medium containing a cell suspension) to eliminate menisci and bubbles that may produce local oxygen gradients. Eliminating the meniscus can reduce or eliminate imaging artifacts and can also reduce mass transfer effects. In addition, by filling the chamber 5, sloshing and the resulting destruction of the chamber contents (e.g., cells or aggregates) can be reduced or eliminated. In addition, by filling the chamber 5 with a liquid (such as a culture medium), the effects of convective circulation can be reduced or eliminated. The absence of bubbles or air spaces above the cell culture can also maximize the utilization of nutrients and growth factors while providing uniform or substantially uniform oxygenation across the entire culture surface area. Finally, by filling the chamber 5 with culture medium, the frequency of replacement of the culture medium can be reduced.

[0103] While filling the chamber 5 provides many advantages, the filling process is not simple. During the fluid introduction and / or removal process, filling can be enhanced by slightly tilting the device 1 (via the second port 25) ( Figure 8 and Fig. 9In fact, if the chamber 5 is in a horizontal position (i.e., perpendicular to gravity) during the introduction and / or removal of fluid, bubbles may form in the chamber 5. In contrast, an inclination of about 1°, about 2°, about 3°, about 4°, about 5°, about 6°, or more may reduce or limit the formation of bubbles during the introduction and / or removal of fluid. In one embodiment, when the bottom wall of the device 1 is supported by an angled surface (such as the cover 60, as described below), the bottom wall of the device 1 is inclined by more than 2°, more than 2.5°, or more than 3°.

[0104] In one embodiment, the bottom wall of the chamber 5 itself can be made to have a slight gradient or angle. In such an embodiment, the top wall of the chamber 5 can also have a gradient or angle accordingly.

[0105] In one embodiment, during fluid introduction and / or removal, the bottom wall of the chamber 5 is tilted or raised at a first corner or edge relative to an opposite second corner or edge. In one embodiment, the second corner or edge is diagonally opposite to the first corner. During fluid introduction, the raised first corner or edge may correspond to the location of the second port 25, and during fluid removal, the raised first corner or edge may correspond to the location of the first port 20.

[0106] In one embodiment, the device 1 may further include a cover 60 ( Fig. 9 and Fig.10 ). The lid 60 can be made to create an incline or angle when the bottom wall of the chamber / container 5 rests on or is supported by the upper plane 62 of the lid 60 (when the lid 60 is on a horizontal surface, for example, a surface in a plane perpendicular to gravity). Thus, the lid 60 can double as a platform to support the device 1, such as during the introduction and / or removal of fluids. As described above, during the introduction and / or removal of fluids, the bottom wall of the chamber / container 5 is inclined when it rests on or is supported by the first corner or edge 63 of the lid 60 and the opposite second corner or edge 64 of the lid 60.

[0107] The cover 60 may include a skirt 65 extending orthogonally downward from the upper plane 62. In one embodiment, the skirt 65 extends continuously around or around the perimeter of the upper plane 62. In one embodiment, the height of the skirt 65 is constant, that is, the skirt 65 extends the same distance orthogonally downward from the upper plane 62 at any point thereof. In one embodiment, the height of the skirt 65 is not constant, that is, the skirt 65 does not extend the same distance orthogonally downward from the upper plane 62.

[0108] In one such embodiment, the height of the skirt 65 is smallest at a first edge or corner 63 of the cover 60, and the height of the skirt 65 is greatest at an opposite second edge or corner 64 of the cover 60. In one embodiment, the height of the skirt 65 changes gradually, traveling from the first edge or corner 63 to the opposite second edge or corner 64 of the cover 60 (in both directions). Thus, when the skirt 65 rests on a horizontal surface (e.g., a surface in a plane perpendicular to gravity), the upper plane 62 of the cover 60 is inclined or angled relative to the surface.

[0109] Regardless of whether the cover 60 covers the housing 3 or supports the housing 3, the first corner or edge 63 and the second corner or edge 64 of the cover 60 are located on a common axis a with the first port 20 and the second port 25. c However, in some embodiments, when the housing 3 is supported by the cover 60 (e.g., when the bottom wall of the container / chamber 5 rests on the upper surface 62 of the cover 60), the bottom wall is tilted about an axis a that is orthogonal to the common axis. t In one embodiment, the tilt axis a t With common axis a c Orthogonal and intersecting. As previously described, the bottom wall is inclined relative to the horizontal surface, and the inclination can be between 0 degrees and 45 degrees. In one embodiment, the inclination is less than 25°. In one embodiment, the inclination is less than 15°. In one embodiment, the inclination is less than 10°. In one embodiment, the inclination is less than 5°. In one embodiment, the inclination is between about 2° and 5°. In one embodiment, the inclination is within the range of about 3°±1°.

[0110] In the case of a cover 60 sized to cover a standard ANSI plate: a 1° bevel angle produces a height difference of approximately 2.5 mm of the skirt 65 between the first corner or edge 63 and the second corner or edge 64; a 2° bevel angle produces a height difference of approximately 5 mm of the skirt 65 between the first corner or edge 63 and the second corner or edge 64; and a 3° bevel angle produces a height difference of approximately 7.5 mm of the skirt 65 between the first corner or edge 63 and the second corner or edge 64.

[0111] When the cover 60 is a hood or cap, the lower side of the upper plane 62 and / or skirt 65 can rest on one or more restrictions 70. In one embodiment, the one or more restrictions 70 are located around the periphery of the one or more side walls 7 (on the opposite side of the one or more side walls 7 relative to where the shoulder 12 is located). In embodiments where the one or more restrictions 70 are located around the periphery of the one or more side walls 7, the one or more restrictions 70 can be continuous around the periphery or can be multiple different elements. In one embodiment, the one or more restrictions are the vertices (or edges) of the one or more side walls 7, and the lower side of the upper plane 62 can rest thereon. In one embodiment, the cover 60 is supported by the vertices of the one or more side walls 7 and continuous or multiple different features located around the periphery of the one or more side walls 7 (as previously described).

[0112] In one embodiment, the height of one or more restrictions 70 (such as second shoulder 72) is not constant, while the height of one or more sidewalls 7 may or may not be constant. In such an embodiment, the height of one or more restrictions 70 is greatest at a first corner or edge 73 of the housing 3, while the height of one or more restrictions 70 is smallest at an opposite second corner or edge 74 of the housing 3. In one embodiment, the height of one or more restrictions 70 gradually decreases from the first corner or edge of the housing 3 to the second corner or edge of the housing 3 (in both directions).

[0113] In one embodiment, the contour of the skirt 65 is complementary to the contour of the one or more restrictions 70. In one embodiment, the contour of the apex (or edge) of the one or more side walls 7 is complementary to the contour of the lower side of the upper plane 62. In one embodiment, the contour of the skirt 65 is complementary to the contour of the one or more restrictions 70, and the bottom wall of the container 5 and the upper plane of the lid 60 (when the skirt 65 rests on the one or more restrictions 70) are both located in parallel planes perpendicular to gravity. In other words, when the container 5 is placed on a horizontal surface, the bottom wall of the container 5 is horizontal or substantially horizontal, and when the skirt 65 rests on the one or more restrictions 70, the upper plane of the lid 60 is located in a plane parallel to the bottom wall plane. Likewise, in such an embodiment, when the lower side (e.g., bottom wall) of the container 5 is located on the upper plane of the lid 60 and the skirt 65 rests on the horizontal surface, the bottom wall of the container 5 is inclined or deflected relative to the horizontal surface.

[0114] In one embodiment, the device 1 comprises a standard lid 60 and further comprises a base adapter that provides sufficient inclination of the bottom wall of the chamber / container 5 for the introduction or withdrawal of fluids.

[0115] The device 1 may be sterile or sterilizable, such as by autoclaving, irradiation or alcohol treatment.

[0116] The apparatus 1 can withstand centrifugal forces up to about 10,000 x g, about 5,000 x g, or about 2,500 x g.

[0117] In another aspect, the apparatus 1 may include: a) a container 5 having one or more side walls 7 extending upwardly and generally orthogonally from a bottom wall; b) one or more restrictions 70 (e.g., a second shoulder) surrounding or encircling a perimeter of the one or more side walls 7 relative to the container 5; and c) a skirted lid 60, wherein a skirt 65 extends downwardly and orthogonally from an upper planar surface 62 of the lid 60 ( Fig.10 A and / or Fig.10 B).

[0118] In one embodiment, the apparatus 1 comprises a single container. In one embodiment, the apparatus 1 comprises a plurality of containers (eg, 6-well, 12-well, 24-well, 48-well or larger well format microplates).

[0119] The one or more restrictions 70 are substantially as described above. In some embodiments where the one or more restrictions 70 are arranged around the perimeter of the one or more side walls 7, the one or more restrictions 70 extend a shorter distance (e.g., height) from the bottom wall relative to the one or more side walls 7. In some embodiments, the one or more restrictions 70 are continuous features around the perimeter of the one or more side walls 7 and may include a second shoulder 72 that extends away from the one or more side walls 7 orthogonally or substantially orthogonally relative to the interior of the container 5. In one embodiment, the height of the one or more restrictions 70 (such as the second shoulder 72) is not constant, while the height of the one or more side walls 7 may or may not be constant. In such an embodiment, the height of the one or more restrictions 70 is greatest at a first corner or edge 73 of the container 5, and the height of the one or more restrictions 70 at an opposite second corner or edge 74 of the container 5 is smallest. In one embodiment, the height of the one or more restrictions 70 gradually decreases from the first corner or edge of the container 5 to the second corner or edge of the container 5 (in both directions).

[0120] The skirt 65 is substantially as described above. In one embodiment, the skirt 65 is continuous, that is, it defines or extends around the perimeter of the cover 60. The height of the skirt 65 is not particularly limited. However, as described above, the height of the skirt 65 at the first edge or corner 63 of the cover 60 may be the smallest, while the height of the skirt 65 at the opposite second edge or corner 64 of the cover 60 may be the largest.

[0121] In one embodiment, the contour of the skirt 65 is complementary to the contour of the one or more restrictions 70, and the bottom wall of the container 5 and the upper plane of the lid 60 (when the skirt 65 rests on the one or more restrictions 70) are both located in parallel planes that are perpendicular to gravity. In other words, when the container 5 is resting on a horizontal surface, the bottom wall of the container 5 is horizontal or substantially horizontal, and when the skirt 65 rests on the one or more restrictions 70, the upper plane of the lid 60 is located in a plane parallel to the bottom wall plane. Likewise, in such an embodiment, when the lower side (e.g., bottom wall) of the container 5 is located on the upper plane of the lid 60 and the skirt 65 rests on the horizontal surface, the bottom wall of the container 5 is inclined or deflected relative to the horizontal surface.

[0122] As described above, an axis (e.g., a common axis) passing through the first edge or corner and the second edge or corner of the cover 60 defines the slope (or direction of tilt / skew) of the bottom wall of the container 5, and an axis (e.g., a tilt axis) orthogonal to the common axis defines the axis about which the bottom wall is tilted or skewed. Thus, the direction and magnitude (e.g., degree) of the tilt / skew affects the travel of the liquid along the bottom wall of the container 5. The degree of tilt or skew about the tilt axis may be as described above.

[0123] In one embodiment, the device 1 further comprises a gas permeable membrane 15. As described above, and incorporated herein by reference, the gas permeable membrane may be sealingly secured to one or more of the side walls 7.

[0124] As also described above, and incorporated herein by reference, the gas permeable membrane 15 may form the bottom wall of the container 5, or may be spaced apart from the bottom wall and lie in a plane parallel to the plane of the bottom wall.

[0125] As also described above, and incorporated herein by reference, a plurality of micropatterned features having any combination of the described properties may be formed in or on the bottom wall of the container 5 .

[0126] In one embodiment, the device 1 further comprises a first port 20 and an opposing second port 25 , each port being in fluid communication with a chamber 5 formed between the bottom wall and the gas permeable membrane 15 and bounded by one or more side walls 7 .

[0127] As also described above, and incorporated herein by reference, the first port 20 and the second port 25 may possess any combination of the described characteristics, including their relationship to the frame 19 .

[0128] method

[0129] Another aspect of the present disclosure provides methods of using the apparatus 1 disclosed above. Such methods may involve assays using the apparatus 1. Such methods may also involve culturing or incubating cells in a cell culture medium within the apparatus 1. Such methods may additionally involve aggregating and culturing cells within the apparatus 1. In some embodiments, the apparatus 1 disclosed above may be used in methods of culturing, incubating and / or aggregating cells under closed conditions; that is, methods of culturing, incubating and / or aggregating cells without direct contact with an environment external to the apparatus 1.

[0130] The method of culturing / incubating / aggregating cells in the apparatus 1 of the present disclosure will require that cells suspended in a liquid (such as a culture medium) be inoculated into the container / chamber 5. In one embodiment, the cells are inoculated via a port (e.g., the second port 25) that is in fluid communication with the container / chamber 5. In one embodiment, the container / chamber 5 is filled with a liquid, such as a culture medium containing a cell suspension. In one embodiment, the liquid is introduced by removing the lid 60 and placing the container / chamber 5 at a certain angle (such as, by resting the housing 3 on the lid 60, as described above), and then draining the liquid into the container / chamber 5. The same steps can be performed for the extraction of the liquid.

[0131] As described above, when liquid is introduced into the container / chamber 5 via the second port 25 (the first port 20 allows the displaced air to be exhausted), it may be necessary to tilt the device 1. The tilt or deflection angle is not particularly limited, but is preferably between 0 and 45 degrees. In one embodiment, the tilt or deflection angle is less than 25°. In one embodiment, the tilt or deflection angle is less than 20°. In one embodiment, the tilt or deflection angle is less than 15°. In one embodiment, the tilt or deflection angle is less than 10°. In one embodiment, the tilt or deflection angle is between about 0 and 10°, between about 1 and 7°, between about 2 and 6°, or in the range of 3°±1°.

[0132] In one embodiment, when or after the liquid and particles (e.g., cells) suspended therein are introduced into the container / chamber 5, they are allowed to settle on the bottom wall under the effect of gravity. In one embodiment, the device 1 is removed from the inclined configuration and returned to the horizontal configuration during the sedimentation operation.

[0133] Once particles (e.g., cells) suspended in a liquid (e.g., culture medium) are introduced into the container / chamber 5, they should be evenly distributed in the liquid, and the particles / cells are expected to settle on the bottom wall with a substantially uniform density. If the container / chamber 5 is filled with liquid, this will help minimize disruptive fluid forces within the container / chamber 5, which may cause uneven distribution. Therefore, in embodiments where the bottom wall includes multiple micropatterned features 30, each such feature is expected to receive a uniform or substantially uniform number of particles / cells. In this case, after a sufficient incubation period, the particle / cell aggregates that appear are expected to fall within a narrow or tight distribution range of aggregate diameters.

[0134] In one embodiment, more than 60% of the aggregates that occur have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, more than 70% of the aggregates that occur have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, more than 80% of the aggregates that occur have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, more than 85% of the aggregates that occur have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, more than 90% of the aggregates that occur have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, more than 95% of the aggregates that occur have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, more than 97% of the aggregates that occur have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, more than 98% of the aggregates that occur have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, more than 99% of the aggregates that occur have a diameter within + / - 10% of the average aggregate diameter.

[0135] If the cells introduced and settled in the container / chamber 5 are adherent cells, but it is not desired that they adhere to the bottom wall, some type of material or treatment may be applied to at least the bottom wall to prevent adhesion. In one embodiment, the container / chamber 5 may be prepared prior to seeding the cells, such as by applying an anti-adhesion rinse solution sold by STEMCELL Technologies.

[0136] After the cells have been in culture / incubation / aggregation conditions for a sufficient period of time, the contents of the container / chamber 5 can be removed by tilting the device 1 (at an angle as described above, such as in conjunction with the lid 60) and removing the fluid via the second port 25.

[0137] In embodiments where the apparatus 1 includes a plurality of micropatterned features 30, the liquid may be removed (as described above) with minimal or no damage to the contents of the micropatterned features 30. In one embodiment, more than 80% of the liquid in the container / chamber 5 may be removed with minimal or no damage to the contents of the micropatterned features 30. In one embodiment, more than 85% of the liquid in the container / chamber 5 may be removed with minimal or no damage to the contents of the micropatterned features 30. In one embodiment, more than 90% of the liquid in the container / chamber 5 may be removed with minimal or no damage to the contents of the micropatterned features 30. In one embodiment, more than 95% of the liquid in the container / chamber 5 may be removed with minimal or no damage to the contents of the micropatterned features 30. In one embodiment, more than 97% of the liquid in the container / chamber 5 may be removed with minimal or no damage to the contents of the micropatterned features 30. In one embodiment, more than 98% of the liquid in the container / chamber 5 may be removed with minimal or no damage to the contents of the micropatterned features 30.

[0138] Once the liquid is removed from the container / chamber 5, fresh liquid can be added thereto (as described above), or a particle / cell / aggregate collection operation can be performed. In one embodiment, particles / cells / aggregates can be collected from the chamber 5 by adding a resuspension buffer or liquid and agitating the device 1 to resuspend the particles / cells / aggregates. In one embodiment, the device 1 can be inverted and centrifuged to resuspend the particles / cells / aggregates. In one embodiment, a suspension buffer can be added to lift the particles / cells / aggregates from the bottom wall, such as by buoyancy.

[0139] In one embodiment, more than 50% of the particles / cells / aggregates are removed from the container / chamber 5. In one embodiment, more than 60% of the particles / cells / aggregates are removed from the container / chamber 5. In one embodiment, more than 70% of the particles / cells / aggregates are removed from the container / chamber 5. In one embodiment, more than 80% of the particles / cells / aggregates are removed from the container / chamber 5. In one embodiment, more than 90% of the particles / cells / aggregates are removed from the container / chamber 5. In one embodiment, more than 95% of the particles / cells / aggregates are removed from the container / chamber 5. In one embodiment, more than 97% of the particles / cells / aggregates are removed from the container / chamber 5. In one embodiment, more than 98% of the particles / cells / aggregates are removed from the container / chamber 5. In one embodiment, more than 99% of the particles / cells / aggregates are removed from the container / chamber 5.

[0140] The method incorporating apparatus 1 may produce clinical or subclinical quantities of cells / aggregates. In embodiments where apparatus 1 is used to aggregate cells, as described above, the aggregates may exhibit a uniform or substantially uniform size distribution, thereby making them suitable for downstream applications, including cell therapy applications. If only subclinical quantities of cells or aggregates are produced in a single apparatus 1, the method may involve multiple apparatuses 1 to produce clinical quantities of cells / aggregates.

[0141] Another aspect of the present disclosure provides a method of manufacturing the device 1 of the present disclosure. The method will include the steps of forming the housing 3, providing the housing 3, forming and / or providing the gas permeable membrane 7, and assembling the various subassemblies to produce the device 1 of the present disclosure. Optionally, the method may include forming, providing and assembling the frame 19 and / or the cover 60.

[0142] Any known process can be used to form the various subassemblies. In one embodiment, the assembly is thermoformed, such as by liquid injection or liquid molding. In one embodiment, the subassembly is machined or milled.

[0143] In one embodiment, the various subcomponents are made from polymers or plastics.

[0144] In one embodiment, the first port 20 and the second port 25 are formed in a subassembly, such as in the frame 19. In one embodiment, the first port 20 and the second port 25 are formed separately from the subassembly. In the latter embodiment, the subassembly (such as the frame 19 and / or the housing 3) that accommodates each of the first port 20 and the second port 25 may need to be drilled and optionally threaded to accommodate the port(s).

[0145] A sheet containing a plurality of micropatterned features (e.g., microwells) can be formed in a variety of ways, including: pouring PDMS onto a mold and curing at an appropriate temperature (e.g., about 90° C.) for an appropriate amount of time (e.g., about 60 minutes); or by thermal forming, such as by heat embossing, e.g. Fig.11 shown.

[0146] In embodiments where the plurality of micropatterned features 30 are formed in the bottom wall of the non-gas permeable membrane 15 of the chamber 5, they may be formed using any known process, including by liquid / injection molding, stamping, etching, heat embossing, and the like.

[0147] In embodiments where the plurality of micropatterned features 30 are formed in the bottom wall of the non-gas permeable membrane 15 of the chamber 5, they may be formed using any known process, including by thermoforming (eg, molding, heat embossing, etc.).

[0148] In an embodiment of a hot embossing method for forming a plurality of micropatterned features 30 in a polymer, the temperature and pressure used to form the plurality of micropatterned features 30 may depend on the polymer. For example, PS can withstand a temperature of about 90°C and a pressure of 1 MPa, PMP can withstand a temperature of about 150°C and a pressure of 5 MPa, PC can withstand a temperature of about 125°C and a pressure of 1 MPa, and SEBS can withstand a temperature of about 110°C and a pressure of 1 MPa. Nevertheless, an optimization experiment was conducted to change the pressing time from 10 seconds to 2 minutes and the pressure from 3 MPa to 9 MPa. Other optimization experiments were also conducted in which the pressing time was changed from 5 seconds to 30 seconds, the temperature was changed from 120°C to 170°C, and the pressure was changed from 10 MPa to 25 MPa.

[0149] In one embodiment, a pressing time of 5 seconds, a pressure of 17.5 MPa, and a temperature of 170°C or less than 150°C are used.

[0150] In one embodiment, embossing time plays an important role in low pressure embossing (below 9 MPa).

[0151] In one embodiment, the cycle time of the hot embossing process is 15 minutes. In one embodiment where the cooling step is omitted, the cycle time can be reduced to about 15 seconds.

[0152] Although Fig.11 The use of silicone as a gasket is shown, but any suitable gasket may be used, such as PMP or other. In one embodiment, the cooling step may be omitted.

[0153] Once formed and / or provided, the subassemblies of the device 1 and the breathable membrane 15 are assembled. In one embodiment, the subassemblies can be clamped to form a leak-proof seal. In one embodiment, fasteners such as screws or rivets can be used to attach the components. In one embodiment, the components can be welded, such as by ultrasonic welding. In one embodiment, the components can be bonded or adhered, such as by glue, tape or other adhesives. In one embodiment, a combination of any of the foregoing assembly methods can be used.

[0154] As mentioned above, the port may be a separate subassembly requiring further assembly, or it may be formed as a subassembly of the housing.

[0155] Regardless of the means used to assemble and secure the subcomponents of the device 1 to one another, it is important that the device 1 as a whole is biocompatible and non-toxic to cells or biomolecules that may be received through the second port 25 and within the chamber 5 .

[0156] The above-described embodiments of the present disclosure are intended to be illustrative and not limiting in any way. These embodiments may be modified in a variety of ways. The present invention and the present disclosure are intended to encompass all such modifications within their scope as defined by the claims, which should be given a broad interpretation consistent with the entire description.

Claims

1. A laboratory device comprising: a housing having one or more side walls extending substantially orthogonally from the planar member; a gas permeable membrane in sealing engagement with the housing, the gas permeable membrane and the housing forming a container having a chamber defined by a top wall and a bottom wall connected and bounded by the one or more side walls; as well as a first port and an opposing second port, each port being in fluid communication with the chamber, The diameter of the second port is the same as or larger than the diameter of the first port.

2. The laboratory apparatus of claim 1, further comprising a plurality of micropatterned features located in the bottom wall of the chamber.

3. The laboratory device according to claim 2, wherein: The gas permeable membrane forms the bottom wall, and the plurality of micropatterned features are formed in or on the gas permeable membrane.

4. The laboratory device according to claim 2, wherein: The gas permeable membrane forms the top wall, and the planar member forms the bottom wall, and the plurality of micropatterned features are formed in or on the planar member.

5. The laboratory device according to any one of claims 1 to 4, wherein: The first port and the second port extend through the top wall.

6. The laboratory device according to any one of claims 1 to 5, wherein: The first port has a diameter of between about 3 mm and 5 mm.

7. The laboratory device according to any one of claims 1 to 6, wherein: The second port has a diameter of between about 3 mm and 12 mm.

8. The laboratory device according to claim 1, wherein: A diameter of the first port and a diameter of the second port are different.

9. The laboratory device according to any one of claims 1 to 8, further comprising a frame located outside the chamber and overlapping at least a perimeter of the gas permeable membrane.

10. The laboratory device according to claim 9, wherein: The frame includes at least one support against the gas permeable membrane for limiting the stretching of the gas permeable membrane and the increase in the volume of the chamber when the chamber is filled with fluid.

11. Laboratory device according to claim 9 or 10, wherein: The first port and the second port traverse opposing corners or edges of the frame.

12. The laboratory device according to claim 11, wherein: The first port and the second port are defined by mating frame wall portions and connecting wall portions, respectively.

13. The laboratory equipment according to claim 12, wherein a height of the connecting wall portion is lower than a height of the frame wall portion.

14. The laboratory apparatus of any one of claims 1 to 13, further comprising a cover having a continuous skirt extending orthogonally downwardly from an upper plane thereof.

15. The laboratory device according to claim 14, wherein: The height of the skirt is smallest at a first edge or corner of the cover, and the height of the skirt is greatest at an opposing second edge or corner of the cover.

16. The laboratory device according to claim 15, wherein: The first edge or corner of the cover, the second edge or corner of the cover, the first port, and the second port are positioned along a common axis when viewed from above and when the cover is in position covering the housing.

17. The laboratory device according to claim 16, wherein: The bottom wall of the container is inclined when the housing is positioned on the lid and the skirt rests on a horizontal surface.

18. The laboratory device according to any one of claims 17, wherein The bottom wall is inclined about an inclined axis orthogonal to the common axis.

19. Laboratory device according to claim 17 or 18, wherein The bottom wall is inclined between 0 and 45 degrees.

20. The laboratory device according to any one of claims 1 to 19, wherein The breathable membrane and the housing are made of polymers independently selected from PS, PMP, PC, PMMA, silicon, silicone-based or styrene block copolymers.

21. The laboratory device according to any one of claims 1 to 20, wherein The micropatterned features are in the shape of cylinders, inverted cones, inverted truncated cones, inverted pyramids, or inverted truncated pyramids.

22. The laboratory device according to any one of claims 1 to 21, wherein The depth of each micropatterned feature is between about 100 μm and 4 mm.

23. The laboratory device according to any one of claims 1 to 22, wherein Each micropatterned feature has a width or diameter in a plane across its opening of between about 100 μm and 5 mm.

24. The laboratory device according to claim 23, wherein: The aspect ratio of each micropatterned feature is 1.

25. A laboratory device comprising: a container having one or more side walls extending upwardly and generally orthogonally from the bottom wall; one or more restrictions surrounding the one or more side walls, the one or more restrictions extending a non-constant and shorter distance from the bottom wall relative to the one or more side walls; as well as A cover having a continuous skirt extending orthogonally downwardly from an upper plane thereof, the skirt being at a minimum height at a first edge or corner of the cover and at a maximum height at an opposite second edge or corner of the cover, wherein the bottom wall lies in a substantially horizontal plane when the container rests on a horizontal surface, and the upper plane of the lid lies in a plane parallel to the bottom wall when the skirt rests on the one or more restrictions, and Wherein, when the skirt rests on the horizontal surface, the bottom wall is inclined relative to the horizontal surface when the lower side of the container is located on the upper plane of the lid.

26. The laboratory device according to claim 25, wherein: The bottom wall is inclined about an inclined axis that is orthogonal to an axis passing through the first edge or corner and the opposing second edge or corner of the cover.

27. The laboratory device according to claim 26, wherein The bottom wall is inclined between 0 and 45 degrees.

28. Laboratory apparatus according to any one of claims 25 to 27, further comprising a gas permeable membrane sealingly secured to the one or more side walls.

29. The laboratory device according to claim 28, wherein The air-permeable membrane forms the bottom wall.

30. The laboratory device according to claim 28, wherein: The gas permeable membrane is spaced apart from the bottom wall and lies in a plane parallel to the plane of the bottom wall.

31. The laboratory device of any one of claims 25 to 30, comprising a plurality of micropatterned features in the bottom wall of the container.

32. The laboratory device according to claim 31, wherein The micropatterned features are in the shape of cylinders, inverted cones, inverted truncated cones, inverted pyramids, or inverted truncated pyramids.

33. Laboratory device according to claim 31 or 32, wherein: The depth of each micropatterned feature is between about 100 μm and 4 mm.

34. Laboratory device according to any one of claims 31 to 33, wherein Each micropatterned feature has a width or diameter in a plane across its opening of between about 100 μm and 5 mm.

35. The laboratory device according to claim 34, wherein: The aspect ratio of each micropatterned feature is less than 1.

36. The laboratory apparatus of claim 30, further comprising a first port and an opposing second port, each port being in fluid communication with a chamber formed between the bottom wall and the gas permeable membrane and bounded by the one or more side walls.

37. The laboratory device according to claim 36, wherein The diameter of the second port is the same as or larger than the diameter of the first port.

38. The laboratory device of claim 36 or 37, further comprising a frame located outside the chamber and overlapping at least the perimeter of the gas permeable membrane.

39. The laboratory device according to claim 38, wherein The frame includes at least one support against the gas permeable membrane for limiting the stretching of the gas permeable membrane and the increase in the volume of the chamber when the chamber is filled with fluid.

40. The laboratory device according to claim 38 or 39, wherein The first port and the second port traverse opposing corners or edges of the frame.

41. The laboratory device according to claim 40, wherein The first port and the second port are defined by mating frame wall portions and connecting wall portions, respectively.

42. The laboratory device according to claim 41, wherein the height of the connecting wall portion is lower than the height of the frame wall portion.

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