Experimental Apparatus and Related Methods

Abstract

The present disclosure relates to laboratory devices and systems, methods of use, and methods of manufacture. The laboratory devices of the present disclosure include a chamber or receptacle and may also include one or more ports fluidly connected to the chamber. Thus, the laboratory device may be a closed system and therefore may be particularly suitable for cell culture applications. In some embodiments, the laboratory device may include a gas-permeable membrane. Filling and removing liquid from the chamber / receptacle of the laboratory device of the present disclosure may be facilitated by features of a lid or adapter that impart a slope or angle to the chamber.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 405,040, filed September 9, 2022, the entire contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to laboratory devices for culturing, incubating, aggregating, etc. cells. More specifically, the present disclosure relates to laboratory devices for culturing, incubating, or aggregating cells on a large scale. [Background technology]

[0003] Two-dimensional (2D) culture of adherent cells in monolayer sheets, such as using T-flasks, is the gold standard. Standard equipment has been developed to allow users to efficiently grow cells in dish or well-plate formats at relatively low cost. In theory, cells grown in 2D monolayers receive uniform amounts of nutrients and growth factors and can easily lift off their growth surface.

[0004] 2D cell culture has several limitations. For example, the formation of a monolayer leads to reduced cell-cell interactions. Furthermore, the plastic surfaces used to support monolayer cultures are much stiffer than the in vivo environment. While the use of hydrogels can mitigate some of the effects of culturing cells on plastic, hydrogels do not completely eliminate this problem.

[0005] In contrast, three-dimensional (3D) culture may be a format that better replicates in vivo conditions during in vitro culture for many cell types. Compared to 2D culture, cells grown in 3D exhibit enhanced cell-cell and cell / extracellular matrix interactions. Improved gene expression, cell junction formation, differentiation, and drug response may be other advantages of certain cell types in 3D culture.

[0006] Nevertheless, current 3D culture systems suffer from several drawbacks, including poor reproducibility, high cost, and increased experimental complexity.

[0007] Various formats for 3D culture are used, including scaffold-based and cell-based assemblies. In scaffold-based assemblies, cells associate with acellular substrates, such as those embedded in hydrogels or porous biomaterials. In cell-based assemblies, cells can spontaneously assemble to form cell aggregates due to cell-cell affinity. 3D culture aggregates have been used in a variety of applications, including expansion, modeling, drug screening, and tissue delivery.

[0008] Given the advantages of 3D culture systems, there is a need to develop cost-effective systems, devices, and methods that reproducibly realize these advantages while overcoming various drawbacks of current systems and devices. Summary of the Invention

[0009] In one aspect of the present disclosure, a laboratory apparatus is provided that may include a housing having one or more side walls extending substantially perpendicularly from a planar member, and a gas permeable membrane in sealing engagement with the housing, the gas permeable membrane and housing being connected by the one or more side walls to form a receptacle having a chamber defined by top and bottom walls surrounded by the one or more side walls.

[0010] The laboratory apparatus of the present disclosure may further include a first port and an opposing second port, each in fluid communication with the chamber. In one embodiment, the first port and the second port are diagonally or diametrically opposed. In one embodiment, the first port and the second port extend through the top wall.

[0011] In one embodiment, the experimental apparatus is closed and / or sealed.

[0012] In one embodiment, the diameter of the second port is the same as or larger than the diameter of the first port. In one embodiment, the diameter of the first port is about 3 mm to 5 mm. In one embodiment, the diameter of the second port is about 3 mm to 12 mm. In one embodiment, the diameter of the first port and the diameter of the second port are not the same.

[0013] The experimental apparatus of the present disclosure may further include a plurality of micropatterned features on the bottom wall of the chamber. In one embodiment, the micropatterned features are cylindrical, inverted conical, inverted truncated conical, inverted pyramidal, or inverted truncated pyramidal. In one embodiment, the depth of each micropatterned feature is about 100 μm to 4 mm. In one embodiment, the width or diameter of each micropatterned feature, taken in a plane intersecting its opening, is about 100 μm to 5 mm. In one embodiment, the aspect ratio of each micropatterned feature is less than 1.

[0014] 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.

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

[0016] The experimental apparatus of the present disclosure may further include a frame external to the chamber and overlapping at least the periphery of the gas-permeable membrane, hi one embodiment, the frame includes at least one brace for the gas-permeable membrane to limit expansion and contraction of the gas-permeable membrane and increase in chamber volume when the chamber is filled with fluid.

[0017] In one embodiment, the first and second ports are formed in and / or extend across opposite corners or edges of the frame.

[0018] In one embodiment, the first port and the second port are bounded by cooperating frame wall portions and connecting wall portions to form first and second port reservoirs, respectively, and in one embodiment, the height of the connecting wall portions is less than the height of the frame wall portions.

[0019] The laboratory apparatus of the present disclosure may further include a lid having a continuous skirt extending perpendicularly downward from its upper surface, hi one embodiment, the skirt has a minimum height at a first edge or corner of the lid and a maximum height at an opposing second edge or corner of the lid.

[0020] In one embodiment, the first edge or corner of the lid, the second edge or corner of the lid, the first port, and the second port lie along a common axis when viewed from above and in a position where the lid is over the housing.

[0021] In one embodiment, the bottom wall of the receptacle is inclined when the housing is placed on the lid and when the skirt is on a horizontal surface. In one embodiment, the bottom wall is inclined about an inclination axis that is perpendicular to the common axis. In one embodiment, the bottom wall is inclined between 0 and 45 degrees, preferably less than 10 degrees, and more preferably 5 degrees or less.

[0022] In one embodiment, at least the frame and the housing are made from a polymer independently selected from polystyrene (PS), polymethylpentene (PMP), polycarbonate (PC), polymethylmethacrylate (PMMA), silicone, silicone-based, or styrene block copolymers. In one embodiment, the gas-permeable membrane is made from polystyrene (PS), polymethylpentene (PMP), polycarbonate (PC), polymethylmethacrylate (PMMA), silicone, silicone-based, or styrene block copolymers.

[0023] Another aspect of the present disclosure provides a laboratory apparatus that may include a receptacle having one or more side walls extending substantially perpendicularly upward from a bottom wall, one or more restrictions surrounding the one or more side walls, the one or more restrictions extending a variable shorter distance from the bottom wall relative to the one or more side walls, and a lid having a continuous skirt extending perpendicularly downward from its upper surface, the skirt having a minimum height at a first edge or corner of the lid and a maximum height at an opposing second edge or corner of the lid.

[0024] In one embodiment, when the receptacle is on a horizontal surface, the bottom wall lies substantially horizontally, and when the skirt rests against the one or more restraints, the top surface of the lid lies in a plane parallel to the bottom wall; and as the skirt rests against the horizontal surface, when the underside of the receptacle is placed against the top surface of the lid, the bottom wall is inclined relative to the horizontal. In one embodiment, the bottom wall is inclined about an inclination axis that is perpendicular to an axis passing through a first edge or corner and an opposing second edge or corner of the lid (as viewed from above). In one embodiment, the bottom wall is inclined between 0 and 45 degrees, preferably less than 10 degrees, and more preferably 5 degrees or less.

[0025] The laboratory apparatus of the present disclosure may further include a gas permeable membrane sealingly secured to one or more side walls.

[0026] In one embodiment, the gas permeable membrane forms the bottom wall.

[0027] In one embodiment, the gas permeable membrane is spaced from and lies in a plane parallel to the plane of the bottom wall (eg, the membrane forms the top wall).

[0028] The experimental device of the present disclosure may further include a plurality of micropatterned features on the bottom wall of the receptacle. In one embodiment, the micropatterned features are cylindrical, inverted conical, inverted truncated conical, inverted pyramidal, or inverted truncated pyramidal. In one embodiment, the depth of each micropatterned feature is about 100 μm to 4 mm. In one embodiment, the width or diameter of each micropatterned feature taken in a plane intersecting its opening is about 100 μm to 5 mm. In one embodiment, the aspect ratio of each micropatterned feature is less than 1.

[0029] The disclosed laboratory apparatus may further include a first port and an opposing second port, each formed between the bottom wall and the gas-permeable membrane and in fluid communication with a chamber surrounded by one or more side walls. In one embodiment, the diameter of the second port is the same as or larger than the diameter of the first port.

[0030] The experimental apparatus of the present disclosure may further include a frame external to the chamber and overlapping at least the periphery of the gas-permeable membrane, hi one embodiment, the frame includes at least one brace for the gas-permeable membrane to limit expansion and contraction of the gas-permeable membrane and increase in chamber volume when the chamber is filled with fluid.

[0031] In one embodiment, the first and second ports extend across and / or are configured at opposite corners or edges of the frame. In one embodiment, the first and second ports are bounded by cooperating frame wall portions and connecting wall portions, respectively, to form first and second port reservoirs. In one embodiment, the height of the connecting wall portions is less than the height of the frame wall portions.

[0032] Another aspect of the present disclosure provides methods of using the disclosed devices in experimental assays, experiments, or incubations. For example, the assays, experiments, or incubations may involve cells or other types of analytes, such as biomolecules. In embodiments involving cells, the methods may involve culturing or incubating the cells, such as to form non-adherent aggregates of cells. Regardless of the process in which the disclosed devices are used, the addition and / or removal of liquid from their receptacles / chambers may be facilitated by tilting the device, such as in cooperation with an included lid. In certain embodiments, the disclosed methods involve closed and / or sealed devices, particularly when the methods involve cells. In such embodiments, liquid (e.g., cell suspension and / or medium) may be introduced into the closed / sealed chamber via a port.

[0033] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments may be put into practice, reference will now be made, by way of example, to the accompanying drawings, which illustrate at least one exemplary embodiment, and which are not intended to limit the scope of the teachings described herein in any way. [Brief explanation of the drawings]

[0034] [Figure 1] 1A-1C show various views of an exemplary device of the present disclosure, including a perspective side view (A), a cross-sectional view (B), and a top view (C). [Figure 2] 1A and 1B show a perspective side view (A) and a cross-sectional view (B) taken from plane "A" of the base / housing of one embodiment of the device of the present disclosure. [Figure 3] 1A and 1B show exploded views of different embodiments of the device of the present disclosure, highlighting potential manufacturing methods. [Figure 4] 1A and 1B show perspective and cross-sectional views of different embodiments of the device of the present disclosure. [Figure 5] 1A-1C illustrate cross-sectional views of various embodiments of micropatterned features. [Figure 6]1A-1C show various embodiments of frames and braces included in the device of the present disclosure (A) and their respective impact on the device chamber volume (B). [Figure 7] Panels (A) and (B) show different embodiments of the port. [Figure 8] 1A-D show images of the fluid collection operation of a device of the present disclosure tilted at either 0° (A), 1° (B), 2° (C), or 3° (D). [Figure 9] The relationship between the base / housing and the lid of an exemplary device is shown in (A) with a partial exploded view of the base / housing floating above the lid. (B) A perspective view, (C) a front view, and (D) a side view of the base / housing tilted above the lid. [Figure 10] 1 illustrates the relationship between the base / housing and lid of an exemplary device. (A) shows a partially exploded view with the lid floating above the base / housing. (B) shows a perspective view and (C) a front view of the lid resting on the base / housing. [Figure 11] 1 illustrates a method of manufacturing a gas-permeable membrane of the present disclosure having a plurality of micropatterned features thermoformed therein. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present disclosure relates to laboratory devices (e.g., cell culture devices), systems, and methods related to their use or manufacture. The devices of the present disclosure can be used to culture, incubate, and / or aggregate cells. In some embodiments, the laboratory device comprises a micropatterned surface (e.g., a surface having multiple microwells). In one embodiment, the laboratory device comprises a closed or sealed chamber. In one embodiment, scaling out beyond the limitations of a single laboratory device (e.g., cell culture device) can be achieved using multiple individual devices.

[0036] As used herein, the term "experimental apparatus" refers to a device used in a laboratory where an experiment or assay can be performed, such as an experiment or assay with a liquid that may contain analytes, biomolecules, or cells. In one embodiment, the experimental apparatus is a cell culture apparatus. Accordingly, as used herein, the term "cell culture apparatus" refers to a device where cells can be seeded and incubated. The cells seeded into the disclosed cell culture apparatus are not particularly limited and can be either adherent or non-adherent cells. Cells placed in the chambers of the disclosed apparatus can be primary cells, cell lines, cancer cells, pluripotent stem cells, or cells differentiated from pluripotent stem cells, etc. In one embodiment, the chambers of the experimental apparatus, particularly at least their surfaces perpendicular to gravity, are not themselves suitable for 2D culture of monolayers of adherent cells. In such embodiments, typically adherent cells seeded into the chamber may rather form suspended cell aggregates, embryoid bodies, or organoids. In one embodiment, at least one surface of the interior chamber or receptacle (perpendicular to gravity) is modified to include multiple micropatterned features (e.g., microwells), as further described below.

[0037] As used in this disclosure, the term "cell aggregate" or "aggregate" refers to a group of cells that have fused to form an interconnected mass. Cells may form aggregates naturally or may be encouraged to form aggregates. Multiple cells may be encouraged to fuse into aggregates when forced into direct contact. Aggregate formation can be influenced by positioning multiple cells relative to surface topology. In embodiments, if the cells are adherent cells, it may be important that their tendency to self-aggregate overcomes their tendency to adhere to non-cellular surfaces, such as cell culture surfaces.

[0038] Unless otherwise defined, scientific and technical terms used in connection with the devices, systems, and methods described herein shall have the meanings commonly understood by those of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0039] Devices and Systems In one aspect of the present disclosure, an experimental device, such as a cell culture device, is provided. In one embodiment, the device of the present disclosure is a closed or sealed system. In other words, the internal chamber of the device is not directly exposed to the external environment, but rather is sealed from the external environment. However, a closed cell culture device may include a gas exchange means for introducing oxygen into the internal chamber. Also, given the need for nutrients and / or growth factors for cells in culture, the closed cell culture device preferably includes a means for introducing nutrients and / or growth factors contained in a cell culture medium into the internal chamber.

[0040] 1 , a device 1 of the present disclosure may include a housing 3 that defines, or cooperates to define, a receptacle and / or chamber 5. The housing 3 may include one or more sidewalls 7 that extend substantially perpendicularly from a substantially planar member 9. In one embodiment, the one or more sidewalls 7 and the planar member 9 are unitary. In one embodiment, the one or more sidewalls 7 and the planar member 9 are of at least two-piece construction.

[0041] 1B and 2, the housing 3 may include a first shoulder 12. The first shoulder 12 extends perpendicularly or substantially perpendicularly away from one or more side walls 7. More specifically, the shoulder 12 may extend from a point midway between the base and the apex of one or more side walls 7 toward the interior of the chamber / receptacle 5. In one embodiment, the shoulder 12 is formed on or in an interior surface of one or more side walls 7 (e.g., a surface of one or more side walls on the chamber / receptacle side). In one embodiment, the shoulder 12 forms a perimeter within the chamber / receptacle 5.

[0042] Shoulder 12 can be any width s w For example, shoulder 12 provides a sufficient surface area for adhesive to be applied, but is not so wide as to significantly reduce the volume of chamber / receptacle 5. In one embodiment, shoulder 12 has a width of about 1 mm. In one embodiment, shoulder 12 has a width of about 2 mm. In one embodiment, shoulder 12 has a width of about 3 mm. In one embodiment, shoulder 12 has a width of about 4 mm. In one embodiment, shoulder 12 has a width of about 5 mm. In a preferred embodiment, shoulder 12 has a width of about 1 mm to 5 mm.

[0043] Shoulder 12 can be set at any height s h The height of the shoulder 12 may be about 2 mm, but should be sufficient to maintain a desired volume within the receptacle / chamber 5. In one embodiment, the height of the shoulder 12 is about 2 mm. The height of the shoulder 12 (taken from the bottom wall) may 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 (taken from the bottom wall) is about 5 mm to 20 mm.

[0044] The housing 3 may 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 materials from which the housing 3 may be made include polystyrene (PS), polymethylpentene (PMP), polycarbonate (PC), polymethylmethacrylate (PMMA), silicon, silicone-based materials, or copolymers such as styrene block copolymers.

[0045] The device 1 may further include a gas-permeable membrane 15. The gas-permeable membrane 15 cooperates with the housing 3 to form a receptacle / chamber 5, which may be defined by a top wall and a bottom wall connected by and surrounded by at least a portion of one or more side walls 7. The gas-permeable membrane 15 may be composed of any material, provided that oxygen and other gases can readily diffuse therethrough (into the receptacle / chamber 5) and that it is non-toxic and does not damage biomolecules or cells or off-gas toxins or contaminants into the receptacle / chamber 5.

[0046] Gas-permeable membrane 15 may be made of any material, but preferably comprises a polymer. In one embodiment, gas-permeable membrane 15 is made of a material suitable for extrusion or molding. Non-limiting examples of materials from which gas-permeable membrane 15 can be made include PS, PMP, PC, SBS / SEBS, silicone, silicone-based materials, or copolymers such as styrene block copolymers.

[0047] In one embodiment, gas-permeable membrane 15 forms the top wall of chamber 5 (as shown in FIGS. 1 and 3); in such cases, the bottom wall of chamber 5 may be planar member 9. Accordingly, gas-permeable membrane 15 may be bonded or otherwise attached to one or more side walls 7, more specifically shoulder 12. The gas-permeable membrane may be otherwise attached to housing 3, such as by any means known to those skilled in the art. In one embodiment, gas-permeable membrane 15 is attached to housing 3 (e.g., one or more side walls 7, or shoulder 12) in a manner that ensures a sealed engagement (e.g., leak-proof) under normal use conditions (e.g., incubation at 37°-75°). Additionally, the choice of adhesive may be important in terms of biocompatibility and / or ability to adhere / bond different materials.

[0048] In the exemplary embodiment shown in FIG. 3A, adhesive 16a is applied to shoulder 12 to secure gas permeable membrane 15 to housing 3. The adhesive can be any type of adhesive, provided it is capable of bonding the materials from which shoulder 12 and gas permeable membrane 15 are made. In one embodiment, the adhesive is double-sided tape. In one embodiment, the adhesive is glue.

[0049] Device 1 may further include frame 17. Frame 17 may provide one or more structural attributes and / or one or more functional attributes. Potential roles of frame 17 may include facilitating fixation of gas-permeable membrane 15 to housing 3 (e.g., shoulder 12), stabilizing gas-permeable membrane 15 from stretching when chamber 5 is filled with liquid, and supporting / incorporating bores or ports through which liquid may be introduced or withdrawn from chamber 5. Thus, in one embodiment, frame 17 may cooperate with housing 3 (e.g., shoulder 12) and adhesives 16a and 16b to attach or fix gas-permeable membrane 15.

[0050] 3B, the gas-permeable membrane 15 is secured to the housing 3 by welding, such as ultrasonic welding. Securing the gas-permeable membrane 15 to the housing 3 by ultrasonic welding may be facilitated by an outer chamber 5 (described further below) of the frame 17 positioned around or overlapping / covering at least the periphery of the gas-permeable membrane 15, such that the membrane 15 is sandwiched between the shoulder 12 and the frame 17. In such an embodiment, the frame 17 and the shoulder 12 may include cooperating ribs 19 that contact and further facilitate ultrasonic welding. In other embodiments, the frame 17 and the shoulder 12 may each include matable ribs and grooves that cooperate to secure the gas-permeable membrane 15 to the housing 3.

[0051] In one embodiment, gas-permeable membrane 15 forms the bottom wall of chamber 5 (as shown in FIG. 4 ); in such a case, the top wall may be planar member 9. Accordingly, gas-permeable membrane 15 may be bonded or otherwise attached to one or more side walls 7. In one embodiment, gas-permeable membrane 15 may be bonded or otherwise attached directly to the edges of one or more side walls 7. In one embodiment, shoulder and / or frame features essentially described above (excluding inversion) may mediate attachment of gas-permeable membrane 15 to housing 3. In one embodiment, gas-permeable membrane 15 is attached to housing 3 in a manner that ensures a sealed engagement (e.g., leak-proof) under normal use conditions; attachment means may be as described above or any other method known to one of skill in the art. Additionally, the choice of adhesive may be important in terms of biocompatibility and / or the ability to adhere / bond dissimilar materials.

[0052] The gas-permeable membrane 15 is not particularly limited in terms of its dimensions, more specifically its thickness, so long as gas can diffuse across it to the same extent, or even better, than the material from which the microplate or cell culture flask is made. In one embodiment, the thickness of the gas-permeable membrane 7 is about 0.05 mm to 1 mm. In one embodiment, the thickness of the gas-permeable membrane 7 is about 0.1 mm to 0.8 mm. In one embodiment, the thickness of the gas-permeable membrane 7 is about 0.15 mm to 0.7 mm. In one embodiment, the thickness of the gas-permeable membrane 7 is about 0.2 mm to 0.65 mm. In one embodiment, the thickness of the gas-permeable membrane 7 is about 0.25 mm to 0.6 mm. In one embodiment, the thickness of the gas-permeable membrane 7 is about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, or thicker.

[0053] In one embodiment, the elements defining, or cooperating to define, chamber 5 may provide different gas permeabilities. In one embodiment, only gas-permeable membrane 15 is permeable to gas or a sufficient amount of gas over the timescale of (cell) culture, incubation, or aggregation. More specifically, housing 3 may not be gas-permeable, or may be permeable only to an insufficient amount / volume of gas over the timescale of (cell) culture, incubation, or aggregation.

[0054] Device 1 may further include at least one port, preferably multiple ports. In one embodiment, device 1 includes first port 20 and second port 25 (FIGS. 1 and 4). First port 20 and second port 25 are each in fluid communication with chamber 5, but may nevertheless be pluggable or pluggable to prevent leakage of contents from chamber 5 and / or to protect the contents of chamber 5 from the environment external to device 1.

[0055] The positioning of the first port 20 and / or the second port 25 may depend on how the chamber 5 is configured with respect to the location of the gas permeable membrane 15. For example, the first port 20 and / or the second port 25 may pass through a top wall of the chamber 5 when the gas permeable membrane 15 forms a bottom wall. As an additional example, the first port 20 and / or the second port 25 may pass through the gas permeable membrane 15, or a respective opening therein, when the membrane forms a top wall of the chamber 5. In one embodiment, the first port 20 and / or the second port 25 may pass through one or more side walls 7.

[0056] In one embodiment, the first port 20 and / or the second port 25 are located on, pass through, or extend through the top wall of the device 1 (e.g., the membrane 15 or the planar member 9, depending on the configuration). Thus, the first port 20 and / or the second port 25 cooperate with their respective bores through the top wall. In one embodiment, one or both ports may extend approximately 0.5-2 mm into the bottom wall. In one embodiment, the first port 20 and / or the second port 25 may be located on, extend through, or pass through one or more side walls 7 of the housing 3.

[0057] The diameter of the first port 20 (and in some embodiments, the diameter of the bore cooperating with the port) does not impede the passage of air therethrough, but is otherwise not particularly limited. In one embodiment, the diameter of the first port 20 (and in some embodiments, the diameter of the bore cooperating with the port) is 3 mm or greater. In one embodiment, the diameter of the first port 20 (and in some embodiments, the diameter of the bore cooperating with the port) is approximately 3 mm to 5 mm. In one embodiment, the diameter of the first port 20 (and in some embodiments, the diameter of the bore cooperating with the port) is approximately 4 mm.

[0058] The diameter of second port 25 (and in some embodiments, the diameter of the bore associated with the port) does not impede the passage of liquids, such as cell culture media. In one embodiment, the diameter of second port 25 (and in some embodiments, the diameter of the bore associated with the port) is the same as or larger than the diameter of first port 20. In one embodiment, the diameter of second port 25 (and in some embodiments, the diameter of the bore associated with the port) is about 3 mm to 12 mm. In one embodiment, the diameter of second port 25 (and in some embodiments, the diameter of the bore associated with the port) is about 10 mm or about 12 mm. A relatively large diameter (e.g., 10 mm or 12 mm) may be preferred when using a serological pipette or similarly sized pipette to introduce and expel fluids into or from chamber 5. When using pipettes smaller than serological pipettes, or using tubing connected to a pump to introduce and expel liquids into and from chamber 5, a relatively small diameter (e.g., 3 mm to 6 mm) may be preferred.

[0059] The ports included in device 1 may be made of any material. Typically, the ports are made of a type of polymer that is amenable to thermoforming. In one embodiment, the ports may be or include a luer fitting.

[0060] In a preferred embodiment, device 1 includes two ports (e.g., first port 20 and second port 25). In such an embodiment, the ports may be located in or near opposite corners or edges of device 1 (e.g., elements 73 and 74 shown in FIGS. 9 and 10). More specifically, the ports may be located diagonally or diametrically in or near opposite corners or edges of device 1, such as on the top wall.

[0061] In one embodiment, the device 1 further includes a plurality of micropatterned features 30 (see FIGS. 4 and 5). The plurality of micropatterned features 30 can be dimensioned to receive a plurality of cells. Thus, in some embodiments, the plurality of micropatterned features 30 are formed or disposed on an inner surface of the chamber 5 that is perpendicular to gravity, such as the bottom wall (e.g., a planar member or a gas-permeable membrane, depending on the configuration). In one embodiment, cells received within each micropatterned feature fuse into (non-attached) aggregates of cells. In one embodiment, the bottom wall of the chamber 5, in or on which the plurality of micropatterned features are formed, does not itself support anchorage-dependent growth of cells.

[0062] In embodiments in which gas-permeable membrane 15 forms the bottom wall of chamber 5, a plurality of micropatterned features 30 may be formed in or on gas-permeable membrane 15. Micropatterned gas-permeable membranes may be manufactured using thermoforming methods such as embossing (see, e.g., FIG. 11 ). In such embodiments, a plurality of micropatterned features 30 may descend from the top surface of gas-permeable membrane 15.

[0063] In embodiments in which gas-permeable membrane 15 forms the top wall of chamber 5, multiple micropatterned features 30 may be formed in or on the bottom wall (e.g., planar member 9). The micropatterned bottom wall may be manufactured using a thermoforming method such as embossing or liquid molding, or by stamping or etching. In such embodiments, multiple micropatterned features 30 may descend from the top surface of the bottom wall (e.g., the base of housing 3).

[0064] Each micropatterned feature may be the same shape. In one embodiment, features of different shapes may be included within the plurality of micropatterned features 30. Nevertheless, the plurality of micropatterned features 30 may be cylindrical, inverted conical, inverted truncated conical, inverted pyramidal, or inverted truncated pyramidal. In a preferred embodiment, the plurality of micropatterned features 30 is an inverted pyramidal or inverted truncated pyramidal (FIG. 5).

[0065] The plurality of micropatterned features 30 can be arranged in any manner, although a more efficient arrangement may be desirable when attempting to maximize the density of micropatterned features on a surface (of a defined surface area). 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 contiguous rows and columns (e.g., a grid when viewed from above).

[0066] In one embodiment, the spacing between adjacent individual features (e.g., microwells) is minimized. Relatively large (i.e., non-minimal) spacing between adjacent individual features (e.g., microwells) can cause inefficiencies when the goal is to maximize the scale of a culture, experiment, or assay. In one embodiment, the spacing between adjacent ones of the plurality of micropatterned features 30 is minimized. In the context of a cell culture device, if the spacing between adjacent individual features is equal to or greater than the diameter of a cell, some cells in chamber 5 may not be deposited within the feature but rather on the spacing. In one embodiment, the protrusion between adjacent individual features (e.g., microwells) is smaller than the diameter of a cell (e.g., <15 μm, <10 μm, <5 μm, <3 μm, <2 μm, or <1 μm). In one embodiment, adjacent ones of the plurality of micropatterned features are separated by an equal pitch.

[0067] In the context of a device 1 used for cell culture, it may be desirable to limit at least the length and width of the device 1 to the ANSI putty format.

[0068] The dimensions of each of the plurality of micropatterned features 30 are not particularly limited. In one embodiment, each of the plurality of micropatterned features 30 is dimensioned to accommodate a plurality of cells. In some embodiments, each of the plurality of micropatterned features 30 is dimensioned to accommodate up to 100 cells. In one embodiment, each of the plurality of micropatterned features 30 is dimensioned to accommodate up to 1000 cells. In one embodiment, each of the plurality of micropatterned features 30 is dimensioned to accommodate up to 5000 cells. In one embodiment, each of the plurality of micropatterned features 30 is dimensioned to accommodate up to 10,000 cells. In one embodiment, each of the plurality of micropatterned features 30 is dimensioned to accommodate more than 10,000 cells.

[0069] In one embodiment, the depth MP of each micropatterned feature d The depth of each micropatterned feature is about 50 μm to 4 mm. In one embodiment, the depth of each micropatterned feature is about 75 μm to 3 mm. In one embodiment, the depth of each micropatterned feature is about 100 μm to 2 mm.

[0070] In one embodiment, the width MP of each micropatterned feature taken in a plane across its opening w Or the diameter is about 50 μm to 5 mm. In one embodiment, the width or diameter of each micropatterned feature taken in a plane across the opening is about 75 μm to 3 mm. In one embodiment, the width or diameter of each micropatterned feature taken in a plane across the opening is about 100 μm to 2 mm.

[0071] In one embodiment, the width of each micropatterned feature taken in a plane across its opening, such as when the micropatterned feature is an inverted pyramid / cone or an inverted frustum of a pyramid / cone, can be about 200 μm, and the depth of such micropatterned features can be about 100-150 μm. In a specific embodiment, the depth of such micropatterned features can be about 140 μm.

[0072] In one embodiment, the width of each micropatterned feature taken in a plane across its opening, such as when the micropatterned feature is an inverted pyramid / cone or an inverted frustum of a pyramid / cone, can be about 400 μm, and the depth of such micropatterned features can be about 250-300 μm. In a specific embodiment, the depth of such micropatterned features can be about 280 μm.

[0073] In one embodiment, the width of each micropatterned feature taken in a plane across its opening, such as when the micropatterned feature is an inverted pyramid / cone or an inverted frustum of a pyramid / cone, can be about 800 μm, and the depth of such micropatterned features can be about 350-400 μm. In a specific embodiment, the depth of such micropatterned features can be about 390 μm.

[0074] MP d and MP w The relationship can be any ratio of the respective dimensions provided herein.

[0075] Notwithstanding the foregoing, the aspect ratio (i.e., the ratio of depth to width across the opening of a micropatterned feature) is not particularly limited. In some embodiments, it may be desirable to minimize disruption of cells or aggregates within the micropatterned features, and therefore, aspect ratios of 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 obtain the contents of each micropatterned feature. In some embodiments, it may be desirable to prioritize recovery of cells or aggregates from within the micropatterned features, and therefore, aspect ratios of about 1 or less may be preferred.

[0076] In one embodiment, the aspect ratio of each micropatterned feature of the plurality of micropatterned features 30 is 1 or less than 1. In one embodiment, the aspect ratio of each micropatterned feature of the plurality of micropatterned features 30 is between 0.5 and 1.

[0077] In embodiments of device 1 including multiple micropatterned features (e.g., microwells), the number of individual such features is not particularly limited. Indeed, the number of individual features is constrained by their dimensions and the dimensions of device 1, particularly its bottom wall. In embodiments, if device 1 occupies a footprint equivalent to or substantially equivalent to a typical microplate (ANSI 1-2004, 127.76 × 85.48 mm), depending on the dimensions, it may be possible to provide over 100,000 individual features. For example, if the width (taken across its opening) of each pyramidal (or truncated) feature is 200 μm, approximately 125,000 individual features can be provided in a single device 1, and if the width (taken across its opening) of each pyramidal (or truncated) feature is 400 μm, approximately 35,000 individual features can be provided in a single device 1.

[0078] 1, 3, 6, and 7, the device 1 may further include a frame 17 of the outer chamber 5 that periphery / covers at least the gas-porous membrane 15. As noted above, the frame 17 may facilitate attachment of the gas-permeable membrane 15 to the housing 3. The frame 17 may also support the gas-porous membrane 15, such as by limiting expansion and contraction of the gas-porous membrane and corresponding increase in chamber volume (when or as the chamber is filled with fluid).

[0079] In one embodiment, frame 17 includes at least one orthosis 40. In one embodiment, at least one orthosis 40 spans opposing or adjacent edges of frame 17 and overlies gas permeable membrane 15 (or underlies gas permeable membrane 15, depending on the configuration of device 1). In one embodiment, device 1 includes a second orthosis 42. In one embodiment, second orthosis 42 spans the same or different opposing or adjacent edges of frame 17 and overlies gas permeable membrane 15 (or underlies gas permeable membrane 15, depending on the configuration of device 1). In one embodiment, second orthosis 42 intersects first orthosis 40. In one embodiment, second orthosis 42 does not intersect first orthosis 40. In embodiments of device 1 that include at least one orthosis (and optionally a second orthosis), the orthosis may support and protect the integrity of gas permeable membrane 7. For example, when gas-permeable membrane 7 forms the upper wall of chamber 5, gas-permeable membrane 7 may foam (expand upward) due to the force of liquid filling chamber 5, thereby creating localized liquid (e.g., cell culture medium) height differences across chamber 5 and potentially creating uneven gradients of waste, oxygen, nutrients, and / or growth factors. Indeed, Figure 7 shows that as the number of appliances decreases, the volume of liquid within chamber 5 increases.

[0080] Frame 17 may be made of any material, but preferably comprises a polymer. In one embodiment, frame 17 is made of a material suitable for molding techniques such as injection molding. Non-limiting examples of materials from which frame 17 may be made include PS, PMP, PC, PMMA, SBS / SEBS, silicone, silicone-based materials, or copolymers such as styrene block copolymers.

[0081] 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 compared to the frame 17 and the housing 3. In one embodiment, each of the membrane 15, the frame 17, and the housing 3 are made of a different material.

[0082] In one embodiment, frame 17 and / or braces 40, 42 are attached to gas permeable membrane 15 using an adhesive (e.g., adhesive 16b). The adhesive can be any type of adhesive, provided it is capable of bonding the materials of frame 17 (and / or brace) and gas permeable membrane 15. In one embodiment, the adhesive is double-sided tape. In one embodiment, the adhesive is glue. In one embodiment, the adhesive used to attach frame 17 and gas permeable membrane 15 is the same adhesive used to attach shoulder 12 and gas permeable membrane 15.

[0083] The dimensions or thickness of the at least one first attachment 40 (and second attachment 42, if present) relative to the gas permeable membrane 15 can affect the diffusion of oxygen therethrough, or the distribution of the diffused oxygen throughout the chamber 5. Generally, a more uniform distribution of oxygen is observed when the surface thickness of the at least one first attachment 40 (and second attachment 42, if present) relative to the gas permeable membrane 15 is on the millimeter scale (e.g., 1-10 mm).

[0084] In certain embodiments of device 1, first port 20 and second port 25 may be configured within or integral with frame 17 and therefore may traverse frame 17. In one embodiment, first port 20 and second port 25 are located at (and traverse) diagonally opposite corners or edges of frame 17 (FIGS. 1 and 7). In one embodiment, first port 20 and second port 25 are integrally located at (and traverse) diagonally opposite corners or edges of frame 17 (FIGS. 1 and 7). In one embodiment, first port 20 and second port 25 are attached to bores molded into diagonally opposite corners or edges of frame 17 (FIGS. 1 and 7).

[0085] Frame 17 may include a peripheral frame wall 45. In one embodiment, frame wall 45 has the same width as or substantially the same width as shoulder 12. In one embodiment, frame wall 45 has a width that is smaller than shoulder 12. In embodiments in which frame wall 45 has a width that is smaller than shoulder 12, the width deficiency may be compensated for by a flange connected to frame wall 45 and extending orthogonally to overlap at least some or all of the width of shoulder 12.

[0086] When positioned on shoulder 12, frame walls 45 extend from shoulder 12 toward or to the apex of one or more side walls 7. In one embodiment, the height of frame walls 45 extends to the apex of one or more side walls 7 when frame 17 is positioned on shoulder 12. In one embodiment, the height of frame walls 45 does not extend to the apex of one or more side walls 7 when frame 17 is positioned on shoulder 12.

[0087] In embodiments in which first port 20 and second port 25 are configured in or on frame 17, they may be located adjacent opposite edges or corners of frame 17 ( FIG. 7 ). Thus, first port 20 and second port 25 may be at least partially bounded by a portion of frame wall 45, and in such cases, first port 20 and second port 25 may each be completely surrounded by connecting wall 47 that cooperates with a portion of frame wall 45. Thus, first port 20 and second port 25 may be surrounded by frame wall portion 45 and connecting wall 47, respectively, to form port reservoir 49. Port reservoir 49 may serve to confine and / or direct fluid introduced into or removed from chamber 5.

[0088] In one embodiment, the height of connecting wall portion 47 is the same as or substantially the same as the height of frame wall 45. That is, the apex of connecting wall portion 47 is the same as or substantially the same as the apex of frame wall 45. In one embodiment, the height of connecting wall portion 47 is less than or shorter than the height of frame wall portion 45 ( FIG. 7 ). A height of connecting wall portion 47 that is less than or shorter than the height of frame wall portion 45 may advantageously reduce siphoning of liquid from port reservoir 49 or from chamber 5 via port reservoir 49 and out of device 1.

[0089] During use, liquid (e.g., culture medium containing a suspension of cells) can be introduced into chamber 5 via second port 25 (e.g., liquid port). To avoid or limit the formation of air bubbles, first port 20 (e.g., vent port) allows air to evacuate chamber 5 when chamber 5 is displaced by liquid (Figure 8). Therefore, the minimum diameter of first port 20 described above can be selected so that liquid does not move across the bore due to surface tension and create a seal.

[0090] The inventors have also unexpectedly discovered many advantages of the disclosed device 1. For example, in the closed (e.g., sealed) configuration of device 1, chamber 5 can be completely filled with liquid (e.g., culture medium containing a suspension of cells), eliminating meniscuses and air bubbles that can create local oxygen gradients. Eliminating the meniscus can reduce or eliminate imaging artifacts and reduce mass transfer effects. Also, completely filling chamber 5 can reduce or eliminate sloshing and resulting disruption of the chamber contents (e.g., cells or aggregates). Furthermore, completely filling chamber 5 with liquid, such as culture medium, can reduce or eliminate the effects of convective circulation. The absence of air bubbles or airspace above the cell culture can also help maximize the availability of nutrients and growth factors while providing uniform or substantially uniform oxygenation across the entire culture surface area. Finally, completely filling chamber 5 with culture medium can reduce the frequency of medium changes.

[0091] While complete filling of chamber 5 offers many advantages, the filling process is not trivial. Filling (through second port 25) can be enhanced by slightly tilting device 1 during fluid introduction and / or removal (FIGS. 8 and 9). Indeed, if chamber 5 is in a horizontal position (i.e., perpendicular to gravity), air bubbles may form within chamber 5 during fluid introduction and / or removal. In contrast, a tilt of about 1°, about 2°, about 3°, about 4°, about 5°, about 6°, or more reduces or limits air bubble formation during fluid introduction and / or removal. In one embodiment, the bottom wall of device 1, when supported by an angled surface (such as lid 60, described below), is tilted by more than 2°, more than 2.5°, or more than 3°.

[0092] In one embodiment, the bottom wall of chamber 5 itself may be manufactured with a slight slope or angle. In such an embodiment, the top wall of chamber 5 may also be correspondingly sloped or angled.

[0093] In one embodiment, the bottom wall of chamber 5 is sloped or raised at a first corner or edge relative to an opposing second corner or edge during fluid introduction and / or removal. In one embodiment, the second corner or edge is diagonally opposite the first corner. During fluid introduction, the raised first corner or edge may correspond to the location of second port 25, and during fluid removal, the raised first corner or edge may correspond to the location of first port 20.

[0094] In one embodiment, device 1 may further include lid 60 (FIGS. 9 and 10). Lid 60 may be manufactured to provide a slope or angle when the bottom wall of chamber / receptacle 5 rests on or is supported by top surface 62 of lid 60 (when lid 60 is on a surface in a horizontal plane, e.g., a plane perpendicular to gravity). Lid 60 may therefore also serve as a platform for supporting device 1, such as during the introduction and / or removal of fluids. As noted above, the bottom wall of chamber / receptacle 5 slopes when resting against or supported by first corner or edge 63 and opposing second corner or edge 64 of lid 60 during the introduction and / or removal of fluids.

[0095] The lid 60 may include a skirt 65 extending perpendicularly downward from the top surface 62. In one embodiment, the skirt 65 extends continuously along or around the periphery of the top surface 62. In one embodiment, the height of the skirt 65 is constant, i.e., the skirt 65 extends perpendicularly downward from the top surface 62 the same distance at any point thereof. In one embodiment, the height of the skirt 65 is not constant, i.e., the skirt 65 does not extend perpendicularly downward from the top surface 62 the same distance.

[0096] In one such embodiment, the height of the skirt 65 is smallest at a first edge or corner 63 of the lid 60 and greatest at an opposing second edge or corner 64 of the lid 60. In one embodiment, the height of the skirt 65 gradually changes as you go from the first edge or corner 63 to the opposing second edge or corner 64 of the lid 60 (in both directions). Thus, when the skirt 65 lies on a horizontal plane (e.g., a plane perpendicular to gravity), the top surface 62 of the lid 60 is inclined or at an angle relative to this surface.

[0097] Regardless of whether the lid 60 is covering or supporting the housing 3, the first corner or edge 63 and the second corner or edge 64 of the lid 60 are aligned with the first port 20 and the second port 25 along a common axis a. c However, in certain embodiments, when the housing 3 is supported by the lid 60 (e.g., when the bottom wall of the receptacle / chamber 5 rests on the top surface 62 of the lid 60), the bottom wall is inclined or along an inclined axis a that is perpendicular to the common axis. t In one embodiment, the tilt axis a t are perpendicular to each other and have a common axis a c As described above, the bottom wall is inclined relative to the horizontal plane, and the degree of inclination can be between 0 and 45 degrees. In one embodiment, the degree of inclination is less than 25 degrees. In one embodiment, the degree of inclination is less than 15 degrees. In one embodiment, the degree of inclination is less than 10 degrees. In one embodiment, the degree of inclination is less than 5 degrees. In one embodiment, the degree of inclination is between about 2 and 5 degrees. In one embodiment, the degree of inclination is within the range of about 3 degrees ± 1 degree.

[0098] In the context of a lid 60 sized to cover a standard ANSI plate: a 1° bevel angle will result in 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 will result in 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 will result in 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.

[0099] When the lid 60 is a cover or cap, the underside of the top surface 62 and / or skirt 65 can rest against one or more restrictions 70. In one embodiment, the one or more restrictions 70 are located around the periphery of one or more side walls 7 (on the opposite side of one or more side walls 7 from where the shoulder 12 is located). In embodiments where one or more restrictions 70 are located around the periphery of one or more side walls 7, the one or more restrictions 70 may be continuous around the periphery or may be multiple discrete elements. In one embodiment, the one or more restrictions are apexes (or edges) of one or more side walls 7, and the underside of the top surface 62 may rest thereon. In one embodiment, the lid 60 is supported by both the apexes of one or more side walls 7 and by continuous or multiple discrete features located around the periphery of one or more side walls 7 (as described above).

[0100] 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 the one or more restrictions 70 is greatest at a first corner or edge 73 of the housing 3, and the height of the one or more restrictions 70 is least at an opposing second corner or edge 74 of the housing 3. In one embodiment, the height of the one or more restrictions 70 gradually decreases (in both directions) from the first corner or edge of the housing 3 to the second corner or edge of the housing 3.

[0101] In one embodiment, the contour of the skirt 65 is complementary to the contour of the one or more restrictors 70. In one embodiment, the contour of the apex (or edge) of one or more side walls 7 is complementary to the contour of the underside of the top surface 62. In one embodiment, the contour of the skirt 65 is complementary to the contour of the one or more restrictors 70, such that both the bottom wall of the receptacle 5 and the top surface of the lid 60 lie in parallel planes perpendicular to gravity (when the skirt 65 rests against the one or more restrictors 70). In other words, when the receptacle 5 is on a horizontal surface, the bottom wall of the receptacle 5 is in a horizontal or substantially horizontal plane, and when the skirt 65 rests against the one or more restrictors 70, the top surface of the lid 60 is in a plane parallel to the plane of the bottom wall. Also, in such an embodiment, when the underside (e.g., bottom wall) of the receptacle 5 is placed on the upper surface of the lid 60 with the skirt 65 relative to a horizontal plane, the bottom wall of the receptacle 5 is inclined or tilted relative to the horizontal plane.

[0102] In one embodiment, the device 1 includes a standard lid 60 and further includes a base adapter that provides sufficient slope of the bottom wall of the chamber / receptacle 5 for the introduction or withdrawal of fluids.

[0103] The device 1 may be sterile or may be sterilizable, such as by autoclaving, radiation, or treatment with alcohol.

[0104] The device 1 can withstand centrifugal forces of up to about 10,000 x g, about 5,000 x g, or about 2,500 x g.

[0105] In another aspect, the device 1 may include a) a receptacle 5 having one or more side walls 7 extending substantially perpendicularly upward from the bottom wall, b) one or more restrictions 70 (e.g., second shoulders) surrounding or around the one or more side walls 7 relative to the receptacle 5, and c) a skirted lid 60, wherein the skirt 65 extends perpendicularly downward from the top surface 62 of the lid 60 (FIGS. 10A and / or 10B).

[0106] In one embodiment, device 1 includes a single receptacle. In one embodiment, device 1 includes multiple receptacles (e.g., a 6-, 12-, 24-, 48-, or larger-well format microplate).

[0107] The one or more restrictions 70 are essentially as described above. In some embodiments, where the one or more restrictions 70 are disposed around the one or more side walls 7, the one or more restrictions 70 extend a shorter distance (e.g., height) from the bottom wall than the one or more side walls 7. In some embodiments, the one or more restrictions 70 are a continuous feature around the one or more side walls 7 and may include a second shoulder 72 that extends perpendicularly or substantially perpendicularly away from the one or more side walls 7 to the interior of the receptacle 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 receptacle 5 and the height of the one or more restrictions 70 is least at an opposing second corner or edge 74 of the receptacle 5. In one embodiment, the height of the one or more restrictions 70 gradually decreases (in both directions) from a first corner or edge of the receptacle 5 to a second corner or edge of the receptacle 5 .

[0108] The skirt 65 is essentially as described above. In one embodiment, the skirt 65 is continuous, i.e., it surrounds or extends around the periphery of the lid 60. The height of the skirt 65 is not particularly limited. However, as described above, the height of the skirt 65 at a first edge or corner 63 of the lid 60 may be smallest, and the height of the skirt 65 at an opposing second edge or corner 64 of the lid 60 may be greatest.

[0109] In one embodiment, the contour of the skirt 65 is complementary to the contour of the one or more restrictors 70, and both the bottom wall of the receptacle 5 and the top surface of the lid 60 lie in parallel planes perpendicular to gravity (when the skirt 65 rests against the one or more restrictors 70). In other words, when the receptacle 5 is on a horizontal surface, the bottom wall of the receptacle 5 lies in a horizontal or substantially horizontal plane, and when the skirt 65 rests against the one or more restrictors 70, the top surface of the lid 60 lies in a plane parallel to the plane of the bottom wall. Also, in such an embodiment, when the underside (e.g., bottom wall) of the receptacle 5 with the skirt 65 is placed on the top surface of the lid 60 relative to a horizontal plane, the bottom wall of the receptacle 5 is inclined or inclined relative to the horizontal plane.

[0110] As described above, an axis (e.g., a common axis) passing through a first edge or corner and a second edge or corner of the lid 60 defines the slope (or direction of the slope / sloped surface) of the bottom wall of the receptacle 5, and an axis orthogonal to the common axis (e.g., tilt axis) defines the axis along which the bottom wall slopes or tilts. Thus, the direction and amplitude (e.g., degree) of the slope / sloped surface affect the movement of liquid along the bottom wall of the receptacle 5. The degree of the slope or slope about the tilt axis may be as described above.

[0111] In one embodiment, device 1 further includes 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 side walls 7.

[0112] Also, as noted above and incorporated herein by reference, the gas permeable membrane 15 may form the bottom wall of the receptacle 5 or may be spaced apart from the plane of the bottom wall and may lie in a plane parallel to the plane of the bottom wall.

[0113] Also, as noted above and incorporated herein by reference, multiple micropatterned features having any combination of the described features may be formed in or on the bottom wall of the receptacle 5.

[0114] In one embodiment, the device 1 further includes a first port 20 and an opposing second port 25, each formed between the bottom wall and the gas-permeable membrane 15 and in fluid communication with a chamber 5 surrounded by one or more side walls 7.

[0115] Also, as noted above and incorporated herein by reference, the first port 20 and the second port 25 may have any combination of the described characteristics, including their relationship to the frame 19 .

[0116] method In another aspect of the present disclosure, there is provided a method of using the device 1 as disclosed above. Such a method may relate to an assay using the device 1. Such a method may further relate to culturing or incubating cells in a cell culture medium within the device 1. Such a method may even further relate to aggregating and culturing cells within the device 1. In some embodiments, the device 1 as disclosed above may be used in a method of culturing, incubating, and / or aggregating cells under closed conditions, i.e., without direct contact with the environment outside the device 1.

[0117] The method of culturing / incubating / aggregating cells with the device 1 of the present disclosure requires seeding cells suspended in a liquid, such as culture medium, into the receptacle / chamber 5. In one embodiment, the cells are seeded through a port (e.g., second port 25) in fluid communication with the receptacle / chamber 5. In one embodiment, the receptacle / chamber 5 is completely filled with a liquid, such as culture medium, containing a suspension of cells. In one embodiment, the liquid is introduced by removing the lid 60, positioning the receptacle / chamber 5 at an angle (e.g., by placing the housing 3 on the lid 60, as described above), and draining the liquid within the receptacle / chamber 5. The same steps can be performed for withdrawing the liquid.

[0118] As described above, since liquid is introduced into the receptacle / chamber 5 through the second port 25 (with the first port 20 allowing for evacuation of displaced air), it may be necessary to tilt the device 1. The angle of the slope or inclined surface is not particularly limited, but is preferably between 0 and 45 degrees. In one embodiment, the angle of the slope or inclined surface is less than 25°. In one embodiment, the angle of the slope or inclined surface is less than 20°. In one embodiment, the angle of the slope or inclined surface is less than 15°. In one embodiment, the angle of the slope or inclined surface is less than 10°. In one embodiment, the angle of the slope or inclined surface is within the range of about 0 to 10°, about 1 to 7°, about 2 to 6°, or 3°±1°.

[0119] In one embodiment, when or after the liquid and particles (e.g., cells) suspended therein are introduced into the receptacle / chamber 5, they may settle under gravity against the bottom wall. In one embodiment, the device 1 is removed from the inclined configuration and returned to the horizontal configuration during the settling operation.

[0120] When particles (e.g., cells) suspended in a liquid (e.g., culture medium) are introduced into the receptacle / chamber 5, they should be uniformly dispersed in the liquid, and the particles / cells are expected to settle at a substantially uniform density against the bottom wall. If the receptacle / chamber 5 is completely filled with liquid, this should help minimize disruptive fluid forces within the receptacle / chamber 5 that could result in uneven distribution. Thus, in embodiments in which 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 such cases, after a sufficient incubation period, the particle / cell aggregates that develop are expected to fall within a narrow or tight distribution of aggregate diameters.

[0121] In one embodiment, more than 60% of the aggregates generated have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, more than 70% of the aggregates generated have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, more than 80% of the aggregates generated have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, more than 85% of the aggregates generated have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, more than 90% of the aggregates generated have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, more than 95% of the aggregates generated have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, more than 97% of the aggregates generated have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, more than 98% of the aggregates generated have a diameter within + / - 10% of the average aggregate diameter. In one embodiment, greater than 99% of the aggregates that arise have a diameter within + / - 10% of the average aggregate diameter.

[0122] If the cells introduced and settled into the receptacle / chamber 5 are adherent cells, but it is undesirable for them to adhere to the bottom wall, certain types of materials or treatments may be applied to at least the bottom wall to prevent adhesion. In one embodiment, the receptacle / chamber 5 may be prepared prior to seeding with cells, such as by coating with an anti-adhesion cleaning solution such as that commercialized by STEMCELL Technologies.

[0123] After the cells have been in culture / incubation / aggregation conditions for a sufficient period of time, the contents of the receptacle / chamber 5 can be removed by tilting the device 1 (at the angle described above, such as in concert with the lid 60) and removing the fluid through the second port 25.

[0124] In embodiments in which the apparatus 1 includes a plurality of micropatterned features 30, the liquid may then be removed (as described above) with minimal or no disruption to the contents of the micropatterned features 30. In one embodiment, greater than 80% of the liquid in the receptacle / chamber 5 may be removed with minimal or no disruption to the contents of the micropatterned features 30. In one embodiment, greater than 85% of the liquid in the receptacle / chamber 5 may be removed with minimal or no disruption to the contents of the micropatterned features 30. In one embodiment, greater than 90% of the liquid in the receptacle / chamber 5 may be removed with minimal or no disruption to the contents of the micropatterned features 30. In one embodiment, greater than 95% of the liquid in the receptacle / chamber 5 may be removed with minimal or no disruption to the contents of the micropatterned features 30. In one embodiment, greater than 97% of the liquid in the receptacle / chamber 5 may be removed with minimal or no disruption to the contents of the micropatterned features 30. In one embodiment, greater than 98% of the liquid in the receptacle / chamber 5 can be removed with minimal or no disruption to the contents of the micropatterned features 30 .

[0125] Once the liquid has been removed from the receptacle / chamber 5, fresh liquid may be added thereto (as described above), or a particle / cell / aggregate harvesting operation may be performed. In one embodiment, the particles / cells / aggregates may be harvested 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 may be inverted and centrifuged to resuspend the particles / cells / aggregates. In one embodiment, a resuspension buffer may be added to lift the particles / cells / aggregates from the bottom wall, such as by buoyancy.

[0126] In one embodiment, greater than 50% of the particles / cells / aggregates are recovered from the receptacle / chamber 5. In one embodiment, greater than 60% of the particles / cells / aggregates are recovered from the receptacle / chamber 5. In one embodiment, greater than 70% of the particles / cells / aggregates are recovered from the receptacle / chamber 5. In one embodiment, greater than 80% of the particles / cells / aggregates are recovered from the receptacle / chamber 5. In one embodiment, greater than 90% of the particles / cells / aggregates are recovered from the receptacle / chamber 5. In one embodiment, greater than 95% of the particles / cells / aggregates are recovered from the receptacle / chamber 5. In one embodiment, greater than 97% of the particles / cells / aggregates are recovered from the receptacle / chamber 5. In one embodiment, greater than 98% of the particles / cells / aggregates are recovered from the receptacle / chamber 5. In one embodiment, greater than 99% of the particles / cells / aggregates are recovered from the receptacle / chamber 5.

[0127] Methods incorporating device 1 may produce clinical or subclinical quantities of cells / aggregates. In embodiments in which device 1 is used to aggregate cells, the aggregates may exhibit a uniform or substantially uniform size distribution, useful for downstream applications, including cell therapy applications, as described above. Where a single device 1 produces only subclinical quantities of cells or aggregates, the method may include multiple devices 1 to produce clinical quantities of cells / aggregates.

[0128] In another aspect of the present disclosure, a method of manufacturing the device 1 of the present disclosure is provided. The method includes forming a housing 3, providing a housing 3, forming and / or providing a gas permeable membrane 7, and assembling various subcomponents to manufacture the device 1 of the present disclosure. Optionally, the method may include forming, providing, and assembling a frame 19 and / or a lid 60.

[0129] The various subcomponents may be formed using any known process. In one embodiment, the components are thermoformed, such as by liquid injection or liquid molding. In one embodiment, the subcomponents are machined or rolled.

[0130] In one embodiment, the various subcomponents are made from polymers or plastics.

[0131] In one embodiment, the first port 20 and the second port 25 are formed in a subcomponent, such as in the frame 19. In one embodiment, the first port 20 and the second port 25 are formed separately from the subcomponent. In the latter embodiment, the subcomponent that receives the first port 20 and the second port 25, respectively, such as the frame 19 and / or the housing 3, may need to be drilled and optionally threaded to receive the port(s).

[0132] Sheets containing multiple micropatterned features (e.g., microwells) can be formed in a variety of ways, including pouring PDMS onto a mold and curing it at an appropriate temperature (e.g., about 90°C) for an appropriate time (e.g., about 60 minutes), or by thermoforming, such as hot embossing, as shown in Figure 11.

[0133] In embodiments in which the plurality of micropatterned features 30 are formed on the bottom wall of the chamber 5 that is not the gas permeable membrane 15, they may be formed using any known process, including by liquid / injection molding, stamping, etching, hot embossing, etc.

[0134] In embodiments in which a plurality of micropatterned features 30 are formed on the bottom wall of chamber 5, which is gas permeable membrane 15, they may be formed using any known process, including by thermoforming (e.g., molding, hot embossing, etc.).

[0135] In embodiments of the hot embossing method for forming the plurality of micropatterned features 30 in a polymer, the temperature and pressure used to form the plurality of micropatterned features 30 can depend on the polymer. For example, PS can withstand temperatures of approximately 90°C and pressures of 1 MPa, PMP can withstand temperatures of approximately 150°C and pressures of 5 MPa, PC can withstand temperatures of approximately 125°C and pressures of 1 MPa, and SEBS can withstand temperatures of approximately 110°C and pressures of 1 MPa. Nevertheless, optimization experiments were conducted varying the press time from 10 seconds to 2 minutes and the pressure from 3 to 9 MPa. Further optimization experiments were conducted varying the press time from 5 to 30 seconds, the temperature from 120°C to 170°C, and the pressure from 10 to 25 MPa.

[0136] In one embodiment, a press time of 5 seconds, a pressure of 17.5 MPa, and a temperature of 170°C or less than 150°C were used.

[0137] In one embodiment, embossing time plays an important role in low pressure embossing (<9 MPa).

[0138] In one embodiment, the hot embossing process has a cycle time of 15 minutes. In one embodiment, if the cooling step is omitted, the cycle time can be reduced to about 15 seconds.

[0139] 11 shows silicone used as the spacer, but any suitable spacer may be used, such as PMP or other spacer. In one embodiment, the cooling step may be omitted.

[0140] Once formed and / or provided, the subcomponents of device 1 and gas permeable membrane 15 are assembled. In one embodiment, the subcomponents may be clamped to create a leak-proof seal. In one embodiment, the components may be attached using fasteners such as screws or rivets. In one embodiment, the components may be welded, such as by ultrasonic welding. In one embodiment, the components may be bonded or attached, such as by glue, tape, or other adhesive. In one embodiment, a combination of any of the foregoing assembly means may be used.

[0141] As noted above, the port may be a separate subcomponent requiring further assembly, or may be formed within a subcomponent of the housing.

[0142] Regardless of the means used to assemble and secure the subcomponents of device 1 to one another, it may be important that the entire device 1 be biocompatible and non-toxic to cells or biomolecules that may be received within chamber 5 via second port 25.

[0143] The above-described embodiments of the present disclosure are intended to be illustrative and not limiting in any way. The embodiments are susceptible to many modifications. The present invention and this disclosure are intended to encompass all such modifications within their scope, as defined by the claims, which should be accorded the broadest interpretation consistent with the entire description.

Claims

1. An experimental device comprising: a housing having one or more side walls extending substantially perpendicularly from the planar member; a gas permeable membrane in sealing engagement with the housing, the gas permeable membrane and the housing forming a receptacle having a chamber defined by top and bottom walls connected and surrounded by the one or more side walls; a first port and an opposing second port, each in fluid communication with the chamber; The laboratory apparatus wherein the diameter of the second port is the same as or larger than the diameter of the first port.

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

3. 3. The laboratory apparatus of 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. 3. The laboratory apparatus of claim 2, wherein the gas permeable membrane forms the top wall, the planar member forms the bottom wall, and the plurality of micropatterned features are formed in or on the planar member.

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

6. The laboratory apparatus of any one of claims 1 to 5, wherein the diameter of the first port is between about 3mm and 5mm.

7. The laboratory apparatus of any one of claims 1 to 6, wherein the diameter of the second port is between about 3mm and 12mm.

8. 10. The laboratory apparatus of claim 1, wherein the diameter of the first port and the diameter of the second port are not the same.

9. The laboratory apparatus of any one of claims 1 to 8, further comprising a frame external to the chamber and overlapping at least the periphery of the gas permeable membrane.

10. 10. The laboratory apparatus of claim 9, wherein the frame includes at least one brace for the gas permeable membrane to limit expansion and contraction of the gas permeable membrane and increase in chamber volume when the chamber is filled with fluid.

11. 11. The laboratory apparatus of claim 9 or 10, wherein the first port and the second port traverse opposite corners or edges of the frame.

12. 12. The laboratory apparatus of claim 11, wherein the first port and the second port are bounded by cooperating frame wall portions and connecting wall portions, respectively.

13. 13. The laboratory apparatus of claim 12, wherein the height of the connecting wall portion is less than the height of the frame wall portion.

14. 14. The laboratory apparatus of any one of claims 1 to 13, further comprising a lid having a continuous skirt extending perpendicularly downwardly from its upper surface.

15. 15. The laboratory apparatus of claim 14, wherein the skirt height is smallest at a first edge or corner of the lid and largest at an opposing second edge or corner of the lid.

16. 16. The laboratory apparatus of claim 15, wherein the first edge or corner of the lid, the second edge or corner of the lid, the first port, and the second port lie along a common axis when viewed from above and when the lid is in a position over the housing.

17. 17. The laboratory apparatus of claim 16, wherein the bottom wall of the receptacle is inclined when the housing is placed on the lid and when the skirt is on a horizontal surface.

18. 18. The laboratory apparatus of claim 17, wherein the bottom wall is tilted about a tilt axis that is perpendicular to the common axis.

19. 19. The laboratory apparatus of claim 17 or 18, wherein the bottom wall is sloped between 0 and 45 degrees.

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

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

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

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

24. 24. The laboratory apparatus of claim 23, wherein each micropatterned feature has an aspect ratio of 1.

25. An experimental device comprising: a receptacle having one or more side walls extending substantially perpendicularly upward from a bottom wall; one or more restrictions surrounding the one or more side walls, the one or more restrictions extending a variable shorter distance from the bottom wall relative to the one or more side walls; a lid having a continuous skirt extending perpendicularly downward from an upper surface thereof, the skirt having a minimum height at a first edge or corner of the lid and a maximum height at an opposing second edge or corner of the lid; when the receptacle is on a horizontal surface, the bottom wall is in a substantially horizontal plane, and when the skirt rests against the one or more restrictions, the top surface of the lid is in a plane parallel to the bottom wall; The laboratory apparatus, wherein the bottom wall is inclined relative to the horizontal plane when the underside of the receptacle is placed on the top surface of the lid, as the skirt rests against the horizontal plane.

26. 26. The laboratory apparatus of claim 25, wherein the bottom wall is inclined about an inclination axis that is perpendicular to an axis passing through the first edge or corner and the opposing second edge or corner of the lid.

27. 27. The laboratory apparatus of claim 26, wherein the bottom wall slopes between 0 and 45 degrees.

28. 28. The laboratory apparatus of any one of claims 25 to 27, further comprising a gas permeable membrane sealingly secured to said one or more side walls.

29. 30. The laboratory apparatus of claim 28, wherein the gas permeable membrane forms the bottom wall.

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

31. The laboratory apparatus of any one of claims 25 to 30, further comprising a plurality of micropatterned features on the bottom wall of the receptacle.

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

33. 33. The experimental device of claim 31 or 32, wherein the depth of each micropatterned feature is between about 100 μm and 4 mm.

34. 34. The laboratory device of any one of claims 31 to 33, wherein the width or diameter of each micropatterned feature taken in a plane across its opening is between about 100 μm and 5 mm.

35. 35. The laboratory apparatus of claim 34, wherein each micropatterned feature has an aspect ratio of less than 1.

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

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

38. 38. The laboratory apparatus of claim 36 or 37, further comprising a frame external to the chamber and overlapping at least the periphery of the gas permeable membrane.

39. 39. The laboratory apparatus of claim 38, wherein the frame includes at least one brace for the gas permeable membrane to limit expansion and contraction of the gas permeable membrane and increase in chamber volume when the chamber is filled with fluid.

40. 40. The laboratory apparatus of claim 38 or 39, wherein the first port and the second port traverse opposite corners or edges of the frame.

41. 41. The laboratory apparatus of claim 40, wherein the first port and the second port are bounded by cooperating frame wall portions and connecting wall portions, respectively.

42. 42. The laboratory apparatus of claim 41, wherein the height of the connecting wall portions is less than the height of the frame wall portions.

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