Patent Description:
It is known to contain three-dimensional cells in a cell culture vessel. It is also known to culture three-dimensional cells in a cell culture vessel. <CIT> discloses a cell culture vessel comprising a plurality of culturing surfaces, a lid, and a magnetized scraper. <CIT> discloses a culture substrate for spheroid culturing. Document <CIT> discloses a further cell culture vessel for spheroid culturing.

The following presents a simplified summary of the disclosure in order to provide a basic understanding of some exemplary embodiments described in the detailed description.

According to the present invention, there is provided a cell culture vessel as defined in claim <NUM>. According to the present invention there is provided a method as defined in claim <NUM>. The cell culture vessel has microcavities, which may be present in an array, in a substrate, for culturing cells in three-dimensional conformation. In order to grow an array of cells growing in three- dimensional conformation, as spheroids or organoids, it is important to provide a substrate in a cell culture vessel that keeps all of the cells in microcavities. When cells grow in microcavities, they form spheroids, confined to a microcavity, and constrained in size. When cells escape from microcavities, or settle onto surfaces inside the cell culture vessel which are not structured and arranged to force the cells to grow in a desired three-dimensional conformation, cells will grow unconstrained. If cells in a cell culture vessel are able to grow unconstrained, they will form inhomogeneous populations of cells.

For some applications, it is desirable to grow and culture a homogeneous population of spheroids inside the cell culture vessel. For example, it is desirable to perform assays in a homogeneous population of cells to control for changes in cell physiology due to inconsistent cell morphology. And, when cellular therapeutics are the desired result of spheroid culture, homogenous cells are also desired to provide controlled, predictable therapeutic results.

When spheroids touch each other, they tend to conglomerate, forming large, non-uniform cellular structures. Cells can escape from microcavities during media changes, for example, when the flow of media into or out of the vessel creates turbulence and causes spheroids to escape the confines of microcavities. As a result, irregular cell agglomerates can form. The structure of the cell culture vessel is important in providing an array of microcavities chamber that constrain the cells to grow in the desired three-dimensional conformation without dislodging the spheroids from their microcavities and allowing them to form irregular cell structures. In embodiments, the disclosure provides a flange and a channel, forming a moat, which allow the vessel to be gently filled with media without creating undue turbulence.

When the interior of the cell culture vessel has flat surfaces, in addition to microcavities, cells can settle onto the flat surfaces and can grow into structures that are not uniform spheroids, but are non-uniform cellular structures. To ensure that cell culture vessels provide an environment that encourage the culture of uniform spheroids, in embodiments, cell culture vessels having reduced flat surfaces are provided. Moat structures, having an angled flange and a channel are provided as an outer perimeter of the substrate that surrounds the plurality of microcavities. And, these channel or moat structures have reduced flat surfaces in the cell culture area. In some embodiments, a method can include culturing cells in the cell culture vessel.

The above embodiments are exemplary and can be provided alone or in any combination with any one or more embodiments provided herein without departing from the scope of the disclosure. Moreover, it is to be understood that both the foregoing general description and the following detailed description present embodiments of the present disclosure, and are intended to provide an overview or framework for understanding the nature and character of the embodiments as they are described and claimed. The accompanying drawings are included to provide a further understanding of the embodiments, and are incorporated into and constitute a part of this specification. The drawings illustrate various embodiments of the disclosure, and together with the description, serve to explain the principles and operations thereof.

These and other features, embodiments, and advantages of the present disclosure can be further understood when read with reference to the accompanying drawings in which:.

Features will now be described more fully hereinafter with reference to the accompanying drawings in which exemplary embodiments of the disclosure are shown. Whenever possible, the same reference numerals are used throughout the drawings to refer to the same or like parts. However, this disclosure can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

A cell culture vessel (e.g., flask) can provide a sterile cell culture chamber for culturing cells. In some embodiments, culturing cells can provide information related to the study of diseases and toxicology, the efficacy of medications and treatments, characteristics of tumors, organisms, genetics, and other scientific, biological, and chemical principles of and relating to cells. The cell culture vessel can include a substrate including a plurality of microcavities (e.g., microcavities, micron-sized wells, submillimeter-sized wells) arranged, for example, in an array. The substrate can be placed in the flask or can form a portion of a boundary wall of the flask. That is, the substrate can be integral to the flask. For example, an array of microcavities can be formed in the bottom interior surface of a cell culture vessel. Or, a substrate having an array of microcavities can be inserted into a cell culture vessel and either rest on the bottom surface of the cell culture vessel or be affixed, by gluing, laser welding, ultrasonic welding, or some other method, to the bottom surface of the cell culture vessel. The substrate can include top and/or bottom sides that include undulating (e.g., sinusoidal) surfaces that form the plurality of microcavities. In some embodiments, the flask can be filled with a material (e.g., media, solid, liquid, gas) that facilitates growth of three-dimensional cell cultures (e.g., cell aggregates, spheroids). For example, a media including cells suspended in a liquid can be added to the cell culture chamber of the vessel. The suspended cells can collect in the plurality of microcavities and can form (e.g., grow) into groups or clusters of cells. These groups or clusters are spheroids or organoids.

For example, in some embodiments, a single spheroid can form in each microcavity of the plurality of microcavities based at least on gravity causing one or more cells suspended in a liquid to fall through the liquid and become deposited within each microcavity. The shape of the microcavity (e.g., a concave surface defining a well), and a surface coating of the microcavity that prevents the cells from attaching to the surface can also facilitate growth of three-dimensional cell cultures in each microcavity. That is, the cells form spheroids and are constrained by the dimensions of the microcavity to grow to a certain size. During culturing, the spheroids can consume media (e.g., food, nutrients) and produce metabolite (e.g., waste) as a byproduct. Thus, in some embodiments food media can be added to the cell culture chamber during culturing and waste media can be removed from the cell culture chamber during culturing. Attempts can be made when adding and removing media to avoid displacing the spheroids from the microcavities and promote desired cell culturing of the spheroids.

As compared to two-dimensional cell cultures, in some embodiments, three-dimensional cell cultures can produce multicellular structures that are more physiologically accurate and that more realistically represent an environment in which cells can exist and grow in real life applications as compared to simulated conditions in a laboratory. For example, three-dimensional cell cultures have been found to more closely provide a realistic environment simulating "in vivo" (i.e. within the living, in a real-life setting) cell growth; whereas two-dimensional cell-cultures have been found to provide an environment simulating "in vitro" (i.e., within the glass, in a laboratory setting) cell growth that is less representative of a real-life environment occurring outside of a laboratory. By interacting with and observing the properties and behavior of three-dimensional cell cultures, advancements in the understanding of cells relating to, for example, the study of diseases and toxicology, the efficacy of medications and treatments, characteristics of tumors, organisms, genetics, and other scientific, biological, and chemical principles of and relating to cells can be achieved.

Embodiments of an exemplary cell culture vessel <NUM> and methods of culturing cells in the exemplary cell culture vessel <NUM> are described with reference to <FIG>. For example, <FIG> schematically illustrates a side view of a cell culture vessel <NUM> having a top <NUM>, a bottom <NUM>, and endwall <NUM>, a neck <NUM> and an opening or aperture <NUM> shown in <FIG> covered by a cap <NUM>. Each of the top <NUM>, the bottom <NUM>, the endwall <NUM> and the neck have interior surfaces. That is, the top <NUM> has an interior surface <NUM>, the bottom has an interior surface <NUM>, the neck <NUM> has an interior surface <NUM>, the endwall <NUM> has an interior surface <NUM>. The cell culture chamber <NUM> is that area of the vessel contained inside the interior surfaces of the vessel. In embodiments, the interior surface <NUM> of the bottom <NUM> has an array of microcavities <NUM>.

<FIG> are schematic drawings of the area shown by circle "<NUM>" of <FIG>. <FIG> illustrate control vessels which result inhomogeneous cell culture. For example, if there are areas in the cell culture chamber <NUM>, where cells may settle, where there are flat surfaces <NUM>, cells <NUM> can congregate on flat surfaces instead of settling in the microcavities <NUM>, and the cells can form irregular cellular conglomerates <NUM>. For example, if there is a frame around the periphery of the array of microcavities, where the substrate comprising the array of microcavities attaches to the walls of the cell culture chamber <NUM> that is a flat ledge, cells may settle on the flat surfaces instead of settling into microcavities <NUM> as they fall through the media and settle on a surface. Examples of these uncontained irregular cellular conglomerates <NUM> are shown schematically in <FIG>. These cells can also invade neighboring microcavities and disrupt the culture of spheroids <NUM> contained in microcavities <NUM>. In contrast, cells contained in microcavities <NUM> form regular, homogeneous spheroids <NUM> (see <FIG>, for example).

<FIG> are photographs of irregular cellular conglomerations that formed on flat portions of cell culture vessels where these flat portions were present, according to Example <NUM>, discussed below.

Turning back to <FIG>, in some embodiments, the vessel <NUM> can include a cap <NUM> oriented to cover the aperture <NUM> to at least one of seal and block the aperture <NUM>, thereby obstructing a path into the cell culture chamber <NUM> from outside the vessel <NUM> through the aperture <NUM>. For clarity purposes, the cap <NUM> is removed and, therefore, not shown in other drawing figures, although it is to be understood that the cap <NUM> can be provided and selectively added to or removed from the aperture <NUM> of the vessel <NUM>, in some embodiments, without departing from the scope of the disclosure. In some embodiments, the cap <NUM> can include a filter that permits the transfer of gas in to and/or out of the cell culture chamber <NUM> of the vessel <NUM>. For example, in some embodiments, the cap <NUM> can include a gas-permeable filter oriented to regulate a pressure of gas within the cell culture chamber <NUM>, thereby preventing pressurization (e.g., over-pressurization) of the cell culture chamber <NUM> relative to a pressure of the environment (e.g., atmosphere) outside the vessel <NUM>.

As shown in <FIG>, and in <FIG>, which shows an alternate cross-sectional view along line <NUM>-<NUM> of <FIG>, e cell culture vessel <NUM> includes a flange <NUM> surrounding at least a portion of the array of microcavities <NUM>. In <FIG> and <FIG>, the array of microcavities <NUM> (also referred to as "substrate") is made up of individual microcavities <NUM> (see <FIG>). Therefore, unless otherwise noted, it is to be understood that, in embodiments, substrate <NUM> can include one or more features of the microcavities 120a, 120b, 120c (See <FIG>). Additionally, the cell culture vessel <NUM> includes a channel <NUM> surrounding at least a portion of the flange <NUM>. Channel <NUM> has an opening <NUM>. As shown in <FIG>, in some embodiments, the flange <NUM> can surround all the microcavities of the array of microcavities <NUM>. However, in some embodiments, the flange <NUM> can surround less than all (e.g., at least a portion of) the microcavities of the plurality of microcavities <NUM>. Likewise, in some embodiments, the opening <NUM> of the channel <NUM> can surround the entire flange <NUM>; however, in some embodiments, the opening <NUM> of the channel <NUM> can surround less than the entire flange <NUM> (e.g., at least a portion of) the flange <NUM>. That is, the channel <NUM> may be closed in some areas, in embodiments. In some embodiments, the flange <NUM> and/or the channel <NUM> can be formed (e.g., manufactured) as an integral component of cell culture vessel <NUM>. Alternatively, in some embodiments, the flange <NUM> and/or the channel <NUM> can be provided as a separate component that can be attached to the cell culture vessel <NUM> to, for example, retrofit an existing cell culture vessel, thereby providing the existing cell culture vessel with one or more features of the flange <NUM> and the channel <NUM> in accordance with embodiments of the disclosure. In embodiments, the array of microcavities <NUM> is integral to the interior surface <NUM> of the bottom <NUM> of the vessel. As shown in <FIG>, the array of microcavities may be provided by an insert, a separate material introduced to or affixed into the flask. For example, a substrate having an array of microcavities <NUM> can be inserted into a cell culture vessel <NUM> and either rest on the bottom surface of the cell culture vessel or be affixed, by gluing, laser welding, ultrasonic welding, or some other method, to the bottom surface of the cell culture vessel.

<FIG> is a top-down view of the vessel, in embodiments. <FIG> illustrates that, in embodiments, the flange <NUM> surrounds the microcavity array <NUM>, and the channel <NUM> surrounds the flange <NUM>.

<FIG> show an enlarged schematic representation of a portion of the cell culture vessel taken at view <NUM> of <FIG>, from a top-down perspective and from a cross-sectional perspective along line <NUM> of <FIG>) showing a substrate having a plurality of microcavities <NUM> in accordance with embodiments of the disclosure. As shown in <FIG>, each microcavity 120a, 120b, 120c of the plurality of microcavities <NUM> includes a concave surface 121a, 121b, 121c (See <FIG>) defining a well 122a, 122b, 122c. Further, each microcavity 120a, 120b, 120c includes an opening 123a, 123b, 123c (e.g., in a first side <NUM> of the substrate <NUM>) to allow liquid and cells to enter the microwells t 122a, 122b, 122c. As shown in <FIG>, in some embodiments, the first side <NUM> of the substrate <NUM> can include a non-linear (e.g., undulating, sinusoidal) profile and a second side <NUM> of the substrate <NUM> can include a planar (e.g., flat) profile. Similarly, as shown in <FIG>, in some embodiments, both the first side <NUM> and the second side <NUM> of the substrate <NUM> can include a non-planar (e.g., undulating, sinusoidal) profile.

Comparing the substrate <NUM> shown in <FIG>, where the first side <NUM> of the substrate <NUM> includes a non-linear (e.g., undulating, sinusoidal) profile and the second side <NUM> of the substrate <NUM> includes a planar (e.g., flat) profile, to the substrate <NUM> shown in <FIG>, where both the first side <NUM> and the second side <NUM> of the substrate <NUM> include a non-planar (e.g., undulating, sinusoidal) profile, it can be seen that, in some embodiments, a thickness of the substrate <NUM>, where both the first side <NUM> and the second side <NUM> of the substrate <NUM> include a non-planar (e.g., undulating, sinusoidal) profile, can be reduced. Thus, in some embodiments, providing both the first side <NUM> and the second side <NUM> of the substrate <NUM> with a non-planar (e.g., undulating, sinusoidal) profile (<FIG>) can reduce the amount of material used to make the substrate <NUM> and can provide a substrate having a microcavity array <NUM> that includes thinner walled microcavities 120a, 120b, 120c than, for example, a substrate having a microcavity array <NUM>, where the first side <NUM> of the substrate <NUM> includes a non-linear (e.g., undulating, sinusoidal) profile and the second side <NUM> of the substrate <NUM> includes a planar (e.g., flat) profile (<FIG>). In some embodiments, thinner walled microcavities 120a, 120b, 120c can permit a higher rate of gas transfer (e.g., permeability) of the substrate to provide more gas in to and out of the well 122a, 122b, 122c during cell culturing. Thus, in some embodiments, providing both the first side <NUM> and the second side <NUM> of the substrate <NUM> with a non-planar (e.g., undulating, sinusoidal) profile (<FIG>) can provide a healthier cell culture environment, thereby improving the culturing of cells in the microcavities 120a, 120b, 120c.

In some embodiments, the substrate <NUM> can include a polymeric material including, but not limited to, polystyrene, polymethylmethacrylate, polyvinyl chloride, polycarbonate, polysulfone, polystyrene copolymers, fluoropolymers, polyesters, polyamides, polystyrene butadiene copolymers, fully hydrogenated styrenic polymers, polycarbonate PDMS copolymers, and polyolefins such as polyethylene, polypropylene, polymethyl pentene, polypropylene copolymers and cyclic olefin copolymers. Additionally, in some embodiments, at least a portion of the well 122a, 122b, 122c defined by the concave surface 121a, 121b, 121c can be coated with an ultra-low binding material, thereby making the at least a portion of the well 122a, 122b, 122c non-adherent to cells. For example, in some embodiments, one or more of perfluorinated polymers, olefins, agarose, non- ionic hydrogels such as polyacrylamides, polyethers such as polyethyleneoxide, polyols such as polyvinylalcohol or mixtures thereof can be applied to at least a portion of the well 122a, 122b, 122c defined by the concave surface 121a, 121b, 121c.

Moreover, in some embodiments, each microcavity 120a, 120b, 120c of the plurality of microcavities <NUM> can include a variety of features and variations of those features without departing from the scope of the disclosure. For example, in some embodiments the plurality of microcavities <NUM> can be arranged in an array including a linear array (shown), a diagonal array, a rectangular array, a circular array, a radial array, a hexagonal close-packed arrangement, etc. Additionally, in some embodiments, the opening 123a, 123b, 123c can include a variety of shapes. In some embodiments, the opening 123a, 123b, 123c can include one or more of a circle, an oval, a rectangle, a quadrilateral, a hexagon, and other polygonal shapes. Additionally, in some embodiments, the opening 123a, 123b, 123c can include a dimension (e.g., diameter, width, diagonal of a square or rectangle, etc.) from about <NUM> microns (µm) to about <NUM>. For example, in some embodiments, the opening 123a, 123b, 123c can include a dimension of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and any dimension or ranges of dimensions encompassed within the range of from about <NUM> to about <NUM>.

In some embodiments, the well 122a, 122b, 122c defined by the concave surface 121a, 121b, 121c can include a variety of shapes. In some embodiments, the well 122a, 122b, 122c defined by the concave surface 121a, 121b, 121c can include one or more of a circular, elliptical, parabolic, hyperbolic, chevron, sloped, or other cross-sectional profile shape. Additionally, in some embodiments, a depth of the well 122a, 122b, 122c (e.g., depth from a plane defined by the opening 123a, 123b, 123c to the concave surface 121a, 121b, 121c can include a dimension from about <NUM> microns (µm) to about <NUM>. For example, in some embodiments, the depth of the well 122a, 122b, 122c can include a dimension of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, any dimension or ranges of dimensions encompassed within the range of from about <NUM> to about <NUM>.

In some embodiments, three-dimensional cells <NUM> (e.g., spheroids, organoids 150a, 150b, 150c) that can be cultured in at least one microcavity 120a, 120b, 120c of the plurality of microcavities <NUM> can include a dimension (e.g., diameter) of from about <NUM> to about <NUM>, and any dimension or ranges of dimensions encompassed within the range of from about <NUM> to about <NUM>. In some embodiments, dimensions greater than or less than the explicit dimensions disclosed can be provided and, therefore, unless otherwise noted, dimensions greater than or less than the explicit dimensions disclosed are considered to be within the scope of the disclosure. For example, in some embodiments, one or more dimensions of the opening 123a, 123b, 123c, the depth of the well 122a, 122b, 122c, and the dimension of the three-dimensional cells <NUM> (e.g., spheroids 150a, 150b, 150c) can be greater than or less than the explicit dimensions disclosed without departing from the scope of the disclosure.

The enlarged view of the cell culture vessel <NUM> at area "<NUM>" of <FIG> including exemplary portions of the flange <NUM> and the channel <NUM>, are shown in <FIG>. For example, in some embodiments, the flange <NUM> can include an inner face <NUM> extending from the substrate <NUM> in a direction away from the concave surface 121a, 121b, 121c (See <FIG>) of each microcavity 120a, 120b, 120c of the plurality of microcavities <NUM> to a perimeter <NUM> of the opening <NUM> of the channel <NUM>. Turning back to <FIG>, in some embodiments, the inner face <NUM> can surround all the microcavities of the plurality of microcavities <NUM>; however, in some embodiments, the inner face <NUM> can surround less than all (e.g., at least a portion of) the microcavities of the plurality of microcavities <NUM>. Likewise, in some embodiments, the opening <NUM> of the channel <NUM> can surround the entire inner face <NUM>; however, in some embodiments, the opening <NUM> of the channel <NUM> can surround less than the entire (e.g., at least a portion of) the inner face <NUM>.

As shown in <FIG>, in some embodiments, the perimeter <NUM> of the opening <NUM> of the channel <NUM> can be spaced a distance "d8" from the portion <NUM> of the substrate <NUM> in a direction away from the concave surface 121a, 121b, 121c (See <FIG>) of each microcavity 120a, 120b, 120c of the plurality of microcavities <NUM>. In some embodiments, the distance "d8" can be within a range of from about <NUM> millimeters (mm) to about <NUM>, for example, from about <NUM> to about <NUM>, although other values, (e.g., less than <NUM> or greater than <NUM>) can be provided in other embodiments without departing from the scope of the disclosure. Additionally, in some embodiments, the perimeter <NUM> of the opening <NUM> of the channel <NUM> can be spaced a distance "d9" from the inner surface <NUM> of the wall <NUM> in a direction toward the portion <NUM> of the substrate <NUM>. Thus, in some embodiments, the opening <NUM> of the channel <NUM> can be defined between the inner surface <NUM> of the endwall <NUM> and the perimeter <NUM>. In some embodiments, the channel <NUM> can include an outer face <NUM> extending from the perimeter <NUM> of the opening <NUM> of the channel <NUM> in a direction toward the concave surface 121a, 121b, 121c (See <FIG>) of each microcavity 120a, 120b, 120c of the plurality of microcavities <NUM> to a base <NUM> of the channel <NUM>. As shown in <FIG>, in some embodiments, the outer face <NUM> can surround all the microcavities of the plurality of microcavities <NUM>; however, in some embodiments, the outer face <NUM> can surround less than all (e.g., at least a portion of) the microcavities of the plurality of microcavities <NUM>. Likewise, in some embodiments, the base <NUM> of the channel <NUM> can surround the entire outer face <NUM>; however, in some embodiments, the base <NUM> of the channel <NUM> can surround less than the entire (e.g., at least a portion of) the outer face <NUM>.

A method of culturing cells in the cell culture vessel <NUM> including the flange <NUM> and the channel <NUM> will now be described with reference to <FIG>. For example, as shown in <FIG>, in some embodiments, the method can include containing a predetermined amount of liquid <NUM> in a region <NUM> of the cell culture chamber <NUM> without liquid of the predetermined amount of liquid <NUM> contacting the channel <NUM>. In some embodiments, the region <NUM> can be defined based at least in part by the flange <NUM> and the substrate <NUM>. For example, in some embodiments, the region <NUM> can be defined based at least in part by inner face <NUM> of the flange <NUM> and the portion <NUM> of the substrate <NUM>. In some embodiments, preventing liquid of the predetermined amount of liquid <NUM> from contacting the channel <NUM>, at this stage of the method, can provide several advantages that, for example, facilitate improved culturing of the cells <NUM>. For example, in some embodiments, at least a portion of the predetermined amount of liquid <NUM> can include a culture solution or media including a liquid including solid particles (e.g., cells) suspended in the liquid. Accordingly, in some embodiments, the method can include depositing liquid <NUM> of the predetermined amount of liquid <NUM> (See <FIG>) in at least one microcavity 120a, 120b, 120c of the plurality of microcavities <NUM> and culturing cells <NUM> (e.g., spheroids 150a, 150b, 150c) in the at least one microcavity 120a, 120b, 120c after depositing the liquid <NUM> in the at least one microcavity 120a, 120b, 120c. For illustrative purposes only, the structures of the array of microcavities <NUM> including the plurality of microcavities <NUM> are omitted from <FIG> with the understanding that, unless otherwise noted, in some embodiments, one or more features of the substrate <NUM> (See <FIG>) can be provided alone or in combination with one or more features of the first exemplary cell culture vessel <NUM> without departing from the scope of the disclosure.

Turning back to <FIG>, in some embodiments, the inner face <NUM> of the flange <NUM> can include a vertical orientation (e.g., extending substantially in the direction of gravity); however, in some embodiments, the inner face <NUM> can be inclined relative to the direction of gravity to, for example, direct cells toward the openings 123a, 123b, 123c of the microcavities 120a, 120b, 120c (See <FIG>). Moreover, in some embodiments, the opening 123a of the microcavity 120a, for example, can be positioned to abut the inner face <NUM> of the flange <NUM>. For example, in some embodiments, the opening 123a of the microcavity 120a can be flush with the inner face <NUM> of the flange <NUM> such that cells suspended in a liquid will fall (e.g., based at least on the force of gravity) and/or be directed by the inner face <NUM> into the reservoir 122a of the microcavity 120a without settling on or adhering to a surface of the vessel <NUM>, including but not limited to the base <NUM> of the channel <NUM>.

In some embodiments, cells that settle on or adhere to a surface of the vessel <NUM>, including but not limited to the base <NUM> of the channel <NUM>, can accumulate and grow (e.g., multiply) outside of the microcavities 120a, 120b, 120c causing problems with respect to desired growth of three-dimensional cells within the microcavities 120a, 120b, 120c. For example, in some embodiments, cells that do not fall (based at least on the force of gravity) into the reservoir 122a, 122b, 122c and that accumulate or attach to other surfaces of the vessel <NUM>, including but not limited to the base <NUM> of the channel <NUM>, can grow outside of the reservoir 122a, 122b, 122c and disrupt (e.g., discourage, alter, slow, or prevent) desired growth of three-dimensional cells within the reservoir 122a, 122b, 122c. Similarly, in some embodiments, cells that accumulate or attach to other surfaces of the vessel <NUM>, including but not limited to the base <NUM> of the channel <NUM>, can grow and dislodge three-dimensional cells in the reservoir 122a, 122b, 122c, thereby disrupting or destroying desired growth of three-dimensional cells within the reservoir 122a, 122b, 122c and altering desired size uniformity of the cells. Accordingly, in some embodiments, by containing the predetermined amount of liquid <NUM> in the region <NUM> of the cell culture chamber <NUM> without liquid of the predetermined amount of liquid <NUM> contacting the channel <NUM>, all cells suspended within the liquid can be directed into the reservoirs 122a, 122b, 122c, thus reducing and eliminating problems that can otherwise occur if cells attach to surfaces of the vessel <NUM>, outside the reservoirs 122a, 122b, 122c, including but not limited to the base <NUM> of the channel <NUM>.

Accordingly, in some embodiments, the method can include depositing liquid <NUM> of the predetermined amount of liquid <NUM> (See <FIG>) on the array of microcavities <NUM>, surrounded by flange <NUM>. In this way, at least one microcavity 120a, 120b, 120c of the plurality of microcavities <NUM> in the microcavity array <NUM> and culturing cells <NUM> (e.g., spheroids 150a, 150b, 150c, shown in <FIG>) in the at least one microcavity 120a, 120b, 120c after depositing the liquid <NUM> on the array of microcavities <NUM>. Moreover, in some embodiments, during culturing, the spheroids 150a, 150b, 150c can consume media (e.g., food, nutrients) and produce metabolite (e.g., waste) as a byproduct. Thus, in some embodiments food media can be added to the cell culture chamber <NUM> during culturing and waste media can be removed from the cell culture chamber <NUM> during culturing. As discussed more fully below, in some embodiments, attempts can be made when adding and removing media to avoid displacing the spheroids 150a, 150b, 150c from the microcavities 120a, 120b, 120c and promote desired cell culturing of the spheroids 150a, 150b, 150c.

Moreover, in some embodiments, with respect to a unit area of the substrate <NUM> (e.g., a unit area providing a respective surface on which one or more cells can be cultured), three-dimensional cell culturing can consume more media (e.g., food, nutrients) and produce more media (e.g., waste) as a byproduct than, for example, a comparable two-dimensional cell culture. Thus, in some embodiments, as compared to, for example, a comparable two-dimensional cell culture, three-dimensional cell cultures in accordance with embodiments of the disclosure can include more frequent media exchanges (e.g., addition of food, nutrients and/or removal of waste) for a comparable period of time. In addition or alternatively, in some embodiments, as compared to, for example, a comparable two-dimensional cell culture, three-dimensional cell cultures in accordance with embodiments of the disclosure can include larger media volumes (e.g., consume more food, nutrients and/or produce more waste) for a comparable period of time. Accordingly, in some embodiments, as discussed more fully below, one or more features of the cell culture vessel <NUM> and the methods of culturing cells <NUM> in the cell culture vessel <NUM> can provide advantages with respect to the frequency of media exchanges as well as the volume of media that can be one or more of contained within the cell culture chamber <NUM> of the vessel <NUM>, added to the cell culture chamber <NUM>, and removed from the cell culture chamber <NUM>, thereby providing a desirable, effective environment in which to culture three-dimensional cells.

For example, as shown in <FIG>, in some embodiments, the method can include adding material (e.g., food, nutrients) from outside the vessel <NUM> into the cell culture chamber <NUM> by inserting a dispensing-port <NUM> into the aperture <NUM> and dispensing material <NUM> from the dispensing-port into the opening <NUM> of the channel <NUM>. Additionally, in some embodiments, the method can include adding material (e.g., food, nutrients) from outside the vessel <NUM> into the cell culture chamber <NUM> by inserting a dispensing-port <NUM> into the aperture <NUM> and dispensing material <NUM> from the dispensing-port into the opening <NUM> of the channel <NUM> after containing the predetermined amount of liquid <NUM> in the region <NUM> of the cell culture chamber <NUM> without liquid of the predetermined amount of liquid <NUM> contacting the channel <NUM>. Thus, in some embodiments, the method can include culturing cells <NUM> in at least one microcavity <NUM> while dispensing material <NUM> from the dispensing-port <NUM> into the opening <NUM> of the channel <NUM>.

<FIG> schematically shows the material <NUM> added into the cell culture chamber <NUM> to, for example, provide the cells <NUM> included in the predetermined amount of liquid <NUM> with food media which the cells <NUM> can consume while being cultured. Likewise, as shown in <FIG>, in some embodiments, the method can include removing material (e.g., waste) from the cell culture chamber <NUM> to outside the vessel <NUM> by inserting a collecting-port <NUM> into the aperture <NUM> and collecting material <NUM> from the channel <NUM> with the collecting-port <NUM>. Additionally, in some embodiments, the method can include removing material (e.g., waste) from the cell culture chamber <NUM> by inserting the collecting-port <NUM> into the aperture <NUM> and collecting material <NUM> from the channel <NUM> with the collecting-port <NUM> after adding material (e.g., food, nutrients) into the cell culture chamber <NUM>. Thus, in some embodiments, the method can include culturing cells <NUM> in at least one microcavity of the plurality of microcavities <NUM> (See <FIG>) while collecting material <NUM> from the channel <NUM> with the collecting-port <NUM>.

<FIG> schematically shows material <NUM> removed from the cell culture chamber <NUM> to, for example, remove waste media, which the cells <NUM> can produce while being cultured, from the predetermined amount of liquid <NUM> and the material <NUM>. For example, in some embodiments, during culturing, the cells <NUM> can consume all or some of the food media (e.g., material <NUM>) added to the cell culture chamber <NUM> and produce (e.g., metabolize as a byproduct) all or some of the waste media (e.g., material <NUM>) removed from the cell culture chamber <NUM>. In some embodiments, while culturing cells <NUM> the methods of adding food media <NUM> to the cell culture chamber <NUM> (See <FIG> and <FIG>) and removing waste media <NUM> from the cell culture chamber <NUM> (See <FIG> and <FIG>) can provide a healthy environment in which the cells <NUM> can be cultured. For example, in some embodiments, methods in accordance with embodiments of the disclosure of adding food media <NUM> to the cell culture chamber <NUM> (See <FIG> and <FIG>) and removing waste media <NUM> from the cell culture chamber <NUM> (See <FIG> and <FIG> can, at least in part, increase one or more of the quality, duration, and effectiveness of the cell culturing process as compared to a cell culture environment to which food media is not added or from which waste media is not removed. Likewise, in some embodiments, one or more features of the vessel <NUM> as well as methods in accordance with embodiments of the disclosure of adding food media <NUM> to the cell culture chamber <NUM> (See <FIG> and <FIG>) and removing waste media <NUM> from the cell culture chamber <NUM> (See <FIG> and <FIG>) can, at least in part, be performed more frequently and with greater effectiveness with respect to the volume of media that can be one or more of contained within the cell culture chamber <NUM> of the vessel <NUM>, added to the cell culture chamber <NUM>, and removed from the cell culture chamber <NUM> during the cell culturing process as compared to, for example, a cell culture vessel not including one or more features of the disclosure as well as methods not including one or more steps of the disclosure.

Additionally, in some embodiments, the method can include moving the vessel <NUM> and collecting the material <NUM> from the channel <NUM> with the collecting-port <NUM>. For example, in some embodiments, moving the vessel <NUM> can include at least one of translating and rotating the vessel <NUM> from a first orientation (e.g., the orientation provided in <FIG>) to a second orientation (e.g., the orientation provided in <FIG>). In some embodiments, the first orientation (e.g., the orientation provided in <FIG>) can provide the vessel <NUM> with the axis <NUM> extending substantially perpendicular relative to the direction of gravity "g" although other orientations of the axis <NUM> relative to the direction of gravity "g" can be provided in other embodiments to define the first orientation. Likewise, in some embodiments, the second orientation (e.g., the orientation provided in <FIG>) can provide the vessel <NUM> with the axis <NUM> extending at an angle that is substantially non-perpendicular relative to the direction of gravity "g" although other orientations of the axis <NUM> relative to the direction of gravity "g" can be provided in other embodiments to define the second orientation.

In some embodiments, the first orientation can provide the axis <NUM> at a first angle relative to the direction of gravity "g" that is different than a second angle of the axis <NUM> relative to the direction of gravity "g" provided by the second orientation. Additionally, in some embodiments, the axis <NUM> of the vessel <NUM> can extend substantially perpendicular to the direction of gravity "g" while one or more of containing the predetermined amount of liquid <NUM> in the region <NUM> of the cell culture chamber <NUM> (See <FIG>), while adding material <NUM> to the cell culture chamber <NUM> (See <FIG>), and while removing material <NUM> from the cell culture chamber <NUM> (See <FIG> and <FIG>). For example, in some embodiments, the vessel <NUM> can be placed on, for example, a horizontal surface (not shown) that defines a major surface perpendicular to the direction of gravity "g" with the axis <NUM> of the vessel <NUM> extending substantially parallel to the major surface of the horizontal surface (not shown) relative to the direction of gravity "g". In addition or alternatively, in some embodiments, the vessel <NUM> can be supported (e.g., held, suspended) by one or more structures (e.g., frame, mount, human hand, etc.) with the axis <NUM> extending substantially perpendicular to the direction of gravity "g". Likewise, in some embodiments, the vessel <NUM> can be supported (e.g., held, suspended) by one or more structures (e.g., frame, mount, human hand, etc.) with the axis <NUM> extending at an angle that is substantially non-perpendicular relative to the direction of gravity "g" as a result of, for example, moving the vessel <NUM> (See <FIG>) before and/or while collecting the material <NUM> from the channel <NUM> with the collecting-port <NUM>.

Thus, as shown schematically in <FIG>, in some embodiments, the method can include moving the vessel <NUM> to cause at least a portion of the predetermined amount of liquid <NUM> and the added material <NUM> (including waste material <NUM>) to flow from the region <NUM> over the flange <NUM> (e.g., over the perimeter <NUM> of the opening <NUM> of the channel <NUM>) and deposit in the channel <NUM>. For example, in some embodiments, while collecting the material <NUM> from the channel <NUM> with the collecting-port <NUM> (See <FIG>), at least a portion of the predetermined amount of liquid <NUM> and the added material <NUM> (including waste material <NUM>) may remain within the region <NUM> of the cell culture chamber <NUM> based, at least in part, on the orientation of the vessel <NUM> and the presence of the flange <NUM> preventing at least the portion of the predetermined amount of liquid <NUM> and the added material <NUM> (including waste material <NUM>) within the region <NUM> from flowing into the channel <NUM>. Thus, in some embodiments, by moving the vessel <NUM> to cause at least a portion of the predetermined amount of liquid <NUM> and the added material <NUM> (including waste material <NUM>) to flow from the region <NUM> over the flange <NUM> and deposit in the channel <NUM>, removal of the waste material <NUM> can be controlled and, in some embodiments, increased relative to, for example, a method where the vessel <NUM> is not moved. In some embodiments, the angle of the axis <NUM> relative to the direction of gravity "g" defined by the second orientation of the vessel <NUM> (See <FIG>) can be selected to, for example, cause at least a portion of the predetermined amount of liquid <NUM> and the added material <NUM> (including waste material <NUM>) to flow from the region <NUM> over the flange <NUM> and deposit in the channel <NUM> without causing cells <NUM> being cultured in the plurality of microcavities <NUM> from dislodging, thereby improving the cell culturing process.

Accordingly, in some embodiments, moving the vessel <NUM> in accordance with embodiments of the disclosure to cause at least a portion of the predetermined amount of liquid <NUM> and the added material <NUM> (including waste material <NUM>) to flow from the region <NUM> over the flange <NUM> and deposit in the channel <NUM> as well as collecting the material <NUM> from the channel <NUM> with the collecting-port <NUM> can provide removal of all or at least a greater quantity of waste material <NUM> from the cell culture chamber <NUM>, as compared to other methods, including but not limited to methods where the vessel <NUM> is not moved (e.g., remains stationary). In some embodiments, the orientation of the axis <NUM> of the vessel <NUM> (e.g., relative to the direction of gravity "g") can remain unchanged during a duration of time while collecting the material <NUM> from the channel <NUM> with the collecting-port <NUM>. Alternatively, in some embodiments, the orientation of the axis <NUM> of the vessel <NUM> (e.g., relative to the direction of gravity "g") can change one or more times during a duration of time while collecting the material <NUM> from the channel <NUM> with the collecting-port <NUM>.

Additionally, as shown in <FIG>, in some embodiments, the method can include adding material (e.g., food, nutrients) into the cell culture chamber <NUM> by inserting the dispensing-port <NUM> into the aperture <NUM> and dispensing material <NUM> from the dispensing-port into the opening <NUM> of the channel <NUM> after removing material <NUM> (e.g., waste) from the cell culture chamber <NUM> (See <FIG>). For example, <FIG> schematically shows waste material <NUM> removed from the cell culture chamber <NUM> after collecting the material <NUM> from the channel <NUM> with the collecting-port <NUM> (See <FIG>) and after moving the vessel <NUM> to cause at least a portion of the predetermined amount of liquid <NUM> and the added material <NUM> (including waste material <NUM>) to flow from the region <NUM> over the flange <NUM> and deposit in the channel <NUM> (See <FIG>). In some embodiments, the added material <NUM> can, for example, replenish the cell culture environment with food and nutrients which the cells <NUM> have consumed and/or depleted. Thus, in some embodiments, the material <NUM> and material <NUM> can include a same or similar composition or different compositions depending on, for example, the type of cell culturing being performed in the vessel <NUM>. Moreover, in some embodiments, the methods of adding material <NUM> (e.g., food, nutrients) (See <FIG> and <FIG>), removing material <NUM> (e.g., waste) (See <FIG>), and adding more material <NUM> (e.g., food, nutrients) (See <FIG>) can be performed selectively one time or multiple times, while culturing cells <NUM>, as the spheroids 150a, 150b, 150c continually (e.g., repeatedly) consume media (e.g., food, nutrients) and/or produce metabolite (e.g., waste) as a byproduct during the cell culturing process.

In some embodiments, one or more features of the flange <NUM> and the channel <NUM> can provide advantages when adding and removing media (See <FIG>) to avoid displacing the spheroids 150a, 150b, 150c from the microcavities 120a, 120b, 120c and promote desired cell culturing of the spheroids 150a, 150b, 150c (see <FIG>). For example, <FIG> illustrates an enlarged schematic representation of a portion of the cell culture vessel <NUM> taken at view <NUM> of <FIG> including the method of adding material <NUM> (e.g., food, nutrients) into the cell culture chamber <NUM> by inserting the dispensing-port <NUM> into the aperture <NUM> and dispensing material <NUM> from the dispensing-port <NUM> into the opening <NUM> of the channel <NUM>. Similarly, FIG. <NUM> illustrates an enlarged schematic representation of a portion of the cell culture vessel <NUM> taken at view <NUM> of FIG. <NUM> including the method of removing material <NUM> (e.g., waste) from the cell culture chamber <NUM> by inserting the collecting-port <NUM> into the aperture <NUM> and collecting material <NUM> from the channel <NUM> with the collecting-port <NUM>.

As shown in <FIG>, in some embodiments, the method of adding material <NUM> (e.g., food, nutrients) into the cell culture chamber <NUM> by inserting the dispensing-port <NUM> into the aperture <NUM> and dispensing material <NUM> from the dispensing-port into the opening <NUM> of the channel <NUM> can include obstructing the flow of material <NUM> along a first flow path 185a, 185b. For example, in some embodiments, the obstructing the flow along the first flow path 185a, 185b can include diverting the flow along the first flow path 185a, 185b with the flange <NUM>. In some embodiments, the diverting the flow along the first flow path 185a, 185b with the flange <NUM> can include, for example, filling the channel <NUM> with the material <NUM> along first flow path 185a, and then flowing the material <NUM> along first flow path 185b from the channel <NUM> into the region <NUM> of the cell culture chamber <NUM>. For example, in some embodiments, the material <NUM> can be dispensed from the dispensing-port <NUM> into the opening <NUM> of the channel <NUM> to gradually fill the channel <NUM> with the material <NUM> along first flow path 185a from the base <NUM> of the channel <NUM> to the perimeter <NUM> of the opening <NUM> of the channel <NUM> along the outer face <NUM> of the channel <NUM>. Additionally, in some embodiments, once the channel <NUM> is filled with material <NUM>, the material <NUM> can then flow over the perimeter <NUM> of the opening <NUM> of the channel <NUM> into the region <NUM> of the cell culture chamber <NUM> along first flow path 185b.

Accordingly, in some embodiments, at least a portion of the region <NUM> (defined based at least in part by the inner face <NUM> of the flange <NUM> and the portion <NUM> of the substrate <NUM>) as well as at least a portion of the cell culture chamber <NUM> can gradually fill with the material <NUM> while the material <NUM> is dispensed from the dispensing-port <NUM> into the opening <NUM> of the channel <NUM>. Although described with respect to adding material <NUM> to the cell culture chamber <NUM>, it is to be understood, unless otherwise noted, that at least a portion of the region <NUM> (defined based at least in part by the inner face <NUM> of the flange <NUM> and the portion <NUM> of the substrate <NUM>) as well as at least a portion of the cell culture chamber <NUM> can gradually fill with the material <NUM> while the material <NUM> is dispensed from the dispensing-port <NUM> into the opening <NUM> of the channel <NUM>, as shown in <FIG>.

Likewise, as shown in <FIG> in some embodiments, the method of removing material (e.g., waste) from the cell culture chamber <NUM> by inserting the collecting-port <NUM> into the aperture <NUM> and collecting material <NUM> from the channel <NUM> with the collecting-port <NUM> can include obstructing the flow of material <NUM> along a second flow path 184a, 184b. For example, in some embodiments, the obstructing the flow along the second flow path 184a, 184b can include diverting the flow along the second flow path 184a, 184b with the flange <NUM>. In some embodiments, the diverting the flow along the second flow path 184a, 184b with the flange <NUM> can include, for example, removing the material <NUM> from the channel <NUM> along second flow path 184a, while flowing the material <NUM> along second flow path 184b. In some embodiments, second flow path 184b can extend from the region <NUM> of the cell culture chamber <NUM> into the channel <NUM>. In addition or alternatively, based at least in part on, for example, a volume of material <NUM>, <NUM>, <NUM> contained within the cell culture chamber <NUM>, in some embodiments, second flow path 184b can extend from the cell culture chamber <NUM> (e.g., outside the region <NUM>) into the channel <NUM>.

Accordingly, in some embodiments, the material <NUM> can be collected from the channel <NUM> with the collecting-port <NUM> to gradually remove the material <NUM> from the channel <NUM> along the second flow path 184a, 184b. Additionally, in some embodiments, for example, after moving the vessel <NUM> to cause at least a portion of the predetermined amount of liquid <NUM> and the added material <NUM> (including waste material <NUM>) to flow from the region <NUM> over the flange <NUM> and deposit in the channel <NUM> (See <FIG>), the material <NUM> can be collected from the channel <NUM> with the collecting-port <NUM> to gradually remove the material <NUM> from the channel <NUM> along the second flow path 184a, 184b. For example, material <NUM> can be gradually removed from the channel <NUM> along second flow path 184a from the perimeter <NUM> of the opening <NUM> of the channel <NUM> along the outer face <NUM> of the channel <NUM> to the base <NUM> of the channel <NUM>. Accordingly, in some embodiments, the material <NUM> can be gradually removed from at least a portion of the cell culture chamber <NUM> as well as from at least a portion of the region <NUM> (defined based at least in part by the inner face <NUM> of the flange <NUM> and the portion <NUM> of the substrate <NUM>) while the material <NUM> is collected with the collecting-port <NUM> from the channel <NUM>.

In some embodiments, while culturing cells <NUM> in at least one microcavity 120a, 120b, 120c of the plurality of microcavities <NUM>, obstructing the flow of material <NUM> along the first flow path 185a, 185b with the flange <NUM> and obstructing the flow of material <NUM> along the second flow path 184a, 184b with the flange <NUM> can respectively add and remove material from the cell culture chamber <NUM> of the vessel <NUM> without, for example, interfering with the culturing of the cells <NUM>. For example, as shown in <FIG>, in some embodiments, the dispensing-port <NUM> can add material <NUM> to the cell culture chamber <NUM> by flowing (e.g., dispensing, blowing, aspirating) the material <NUM> from the dispensing-port <NUM> into the channel <NUM> with a velocity along the first flow path 185a, 185b, thereby creating a positive pressure force in and around the channel <NUM>. Likewise, as shown in <FIG>, in some embodiments, the collecting-port <NUM> can remove material <NUM> from the cell culture chamber <NUM> by flowing (e.g., collecting, sucking) the material from the channel <NUM> into the collecting-port <NUM> with a velocity along the second flow path 184a, 184b, thereby creating a negative pressure force in and around the channel <NUM>. Accordingly, in some embodiments, the flange <NUM> can slow a velocity of the material <NUM>, <NUM> respectively flowing along the first flow path 185a, 185b and the second flow path 184a, 184b, thereby respectively decreasing the positive pressure force and the negative pressure force in and around channel <NUM>.

For example, in some embodiments, the method of adding material (e.g., food, nutrients) into the cell culture chamber <NUM> by inserting the dispensing-port <NUM> into the aperture <NUM> and dispensing material <NUM> from the dispensing-port into the opening <NUM> of the channel <NUM> (See <FIG>) can provide a slow, continuous, and controlled (e.g., non-turbulent) flow of material <NUM> into the cell culture chamber <NUM> as compared to, for example, a method or cell culture vessel not including one or more features of the flange <NUM> and the channel <NUM>. Likewise, in some embodiments, the method of removing material (e.g., waste) from the cell culture chamber <NUM> by inserting the collecting-port <NUM> into the aperture <NUM> and collecting material <NUM> from the channel <NUM> (See <FIG>) can provide a slow, continuous, and controlled (e.g., non-turbulent) flow of material <NUM> out of the cell culture chamber <NUM> as compared to, for example, a method or cell culture vessel not including one or more features of the flange <NUM> and the channel <NUM>. By reducing a velocity of the material <NUM>, <NUM> respectively flowing along the first flow path 185a, 185b and the second flow path 184a, 184b, in some embodiments, the flange <NUM> and the channel <NUM> can, therefore, prevent cells <NUM> being cultured in at least one microcavity 120a, 120b, 120c of the plurality of microcavities <NUM> from dislodging. For example, in some embodiments, if flow of material dislodges one or more cells <NUM>, one or more microcavities 120a, 120b, 120c can include more than one spheroid or no spheroids. Accordingly, in some embodiments, by providing a slow, continuous, and controlled (e.g., non-turbulent) flow of material, based at least in part on one or more features of the flange <NUM> and the channel <NUM>, in accordance with methods and embodiments of the disclosure, the likelihood of dislodging cells <NUM> being cultured in the vessel <NUM> can be reduced and better quality cell cultures and more accurate scientific results relating to the cell cultures can be obtained.

HCT116, a colon cancer cell line, was grown to ~<NUM>% confluency in a flask in complete McCoy's 5a media (<NUM>% fetal bovine serum with <NUM> units/mL of penicillin/ 10ug/mL streptomycin) and trypsinized to detach the cells from the surface. The cells were then counted and resuspended in complete McCoy's media at a final concentration of cells at <NUM> cells per microcavity (for T-<NUM> microcavity flask add <NUM> of cells at final concentration of ~<NUM> x <NUM>^<NUM> cells/mL). Allow microcavity flask to sit at room temperature to allow for the cells to settle into the surface of the microcavity flask. After room temperature incubation, place flask in to <NUM> incubator with <NUM>% CO2 and <NUM>% relative humidity for the duration of the experiment. Spheroid formation occurs within the first <NUM> hours.

Control Flask: HTC116 cells were seeded into a T75 flask that was prepared by cutting away the bottom surface of the flask and affixing material having an array of microcarriers to the bottom of the flask. Because the flask was cut away, a "lip" of material remained around the inner periphery of the bottom surface of the flask. This flat surface, around the periphery of the array of microcavities, was present during culture in the control vessel.

Experimental Flask: HTC116 cells were also seeded into a T75 flask having the flange and channel structure illustrated in <FIG> and <FIG>, for example.

After media with suspended cells were added to the inner area, flask was allowed to stay in static conditions for <NUM> at room temperature to allow cells to settle into microwells. This <NUM> incubation step is used in cell culture biology labs to ensure uniform cell seeding and to minimize edge effects upon subsequent placement of the vessels into incubator chamber. After cells settled into the microcavities, an additional amount of cell culture media was then added to the flask. In the control flask, media was added with a pipette to the mid-center of the bottom surface of the flask. In the experimental flask, media was added as shown, for example, in <FIG>. The flask was then placed into cell culture incubator and cultured for <NUM> days. During this culture period total of <NUM> media exchanges, with <NUM>% media exchange efficiency, were performed.

<FIG> is a photograph of spheroids growing in the control T75 flask. Spheroids are irregular and missing from some microcavities (having possibly been dislodged during media changes). In addition, irregular cellular conglomerates were seen in the control flask, such as those shown in <FIG>. <FIG> is a photograph showing spheroids in the experimental flask after <NUM> days of culture. Spheroids appear to be similar in size and all wells are filled with spheroids. <FIG> demonstrates <NUM>% spheroid retention in the flask assembled according to the experimental design with media changes.

Following <NUM> days of culture, spheroids were harvested from the control flask (<FIG>) and from the experimental flask of, for example, <FIG> (<FIG> are photographs of the harvested cells. As is seen in <FIG>, cells formed "ropes" of cells in vessels that had flat surfaces in the cell culture area (see white arrows). Other irregular cellular conglomerates can be seen in <FIG>. In contrast, spheroids grown in the experimental flask according to <FIG> were regular and homogeneous, as shown in <FIG>.

Throughout the disclosure, the terms "material", "liquid", and "gas" can be used to describe properties of a material employed when, for example, culturing cells in the cell culture vessel. Unless otherwise noted, for purposes of the disclosure, "material" can include fluid material (e.g., liquid or gas). Additionally, material can include a culture solution or media including a liquid including solid particles (e.g., cells) suspended in the liquid. Unless otherwise noted, for purposes of the disclosure, "liquid" can include cleaning or rinsing solutions, aqueous solutions, or other liquid that can be added to or removed from the vessel to, for example, clean the cell culture chamber, sterilize one or more features of the substrate and the vessel, prepare the substrate for cellular growth and other uses of liquid. Additionally, liquid can include a culture solution or media including a liquid including solid particles (e.g., cells) suspended in the liquid. Unless otherwise noted, for purposes of the disclosure, "gas" can include air, filtered or treated air, or other gases.

Throughout the disclosure, the terms "non-permeable", "gas-permeable", and "porous" can be used to describe properties (e.g., material properties, characteristics, parameters) of one or more features of a substrate.

Unless otherwise noted, for purposes of the disclosure, a "non-permeable" substrate (e.g., material of a non-permeable substrate) is considered to be impermeable to solid, liquid, and gas under normal conditions (e.g., no external influence including but not limited to pressure and force) and, therefore, does not permit the transfer of solid, liquid, or gas in to, through, or out of, the non-permeable substrate under normal conditions. In some embodiments, a non-permeable substrate can form a portion of the wall of the vessel. Additionally, the cell culture chamber of the vessel is considered to be sterile when a non-permeable substrate forms a portion of the wall of the vessel because bacteria, for example, cannot pass through the non-permeable substrate. However, when filling the plurality of microcavities of the substrate with material, gas can become trapped within the microcavity of a non-permeable substrate based on surface tension of the liquid, thereby, in some embodiments, preventing material from filling the microcavities and preventing growth of a spheroid.

Unless otherwise noted, for purposes of the disclosure, a "gas-permeable" substrate (e.g., material of a gas-permeable substrate) is considered to be impermeable to solid and liquid, and permeable to gas under normal conditions. Therefore, a gas-permeable substrate does not permit the transfer of solid and liquid in to, through, or out of, the gas-permeable substrate and does permit the transfer of gas in to, through, or out of, the gas-permeable substrate. In some embodiments, a gas-permeable substrate can form a portion of the wall of the vessel. Additionally, the cell culture chamber of the vessel is considered to be sterile when a gas-permeable substrate forms a portion of the wall of the vessel because bacteria, for example, cannot reasonably pass through the gas-permeable substrate. However, although the substrate is gas-permeable, gas can still become trapped in the microcavity during filling with material because gas-permeation rates through the gas-permeable substrate can be slower than the rate required to displace gas from the cavity under ordinary operating conditions and can therefore take an unacceptably long amount of time to permeate through the substrate. Thus, in some embodiments, slowly filling the microcavities allows the liquid front to enter each microcavity at an angle, thereby displacing gas as the liquid fills the microcavity. In some embodiments, after filling the cavity with liquid, gas can permeate (slowly) through the gas-permeable substrate.

Unless otherwise noted, for purposes of the disclosure, a "porous" substrate (e.g., material of a porous substrate) is considered to be impermeable to solid and permeable to liquid and gas under normal conditions. Therefore, a porous substrate does not permit the transfer of solid in to, through, or out of, the porous substrate and does permit the transfer of liquid and gas in to, through, or out of, the porous substrate. A porous substrate cannot form a portion of the vessel because bacteria can pass through a porous substrate, thus causing sterility issues in the cell culture chamber. Thus, when using a porous substrate, the substrate must be enclosed (entirely enclosed) in the sterile cell culture chamber of the vessel. During filling of the microcavities with material, however, gas can escape (e.g., pass) through the porous substrate. Thus, filling of the microcavities can be performed rapidly without concern for entrapping gas in the microcavities. In some embodiments, liquid can only pass through the porous substrate with added pressure or physical contact and disturbance of the substrate. Thus, in some embodiments, material including liquid can be contained in the microcavities of the substrate so long as the substrate is not exposed to added pressure or physical contact and disturbance. For example, in some embodiments, the porous substrate can be supported in the cell culture chamber to allow gas to pass through the substrate during filling as well as during culturing and to isolated the substrate from added pressure or physical contact and disturbance from external forces (e.g., outside the cell culture chamber).

It is to be understood that, as used herein the terms "the," "a," or "an," mean "at least one," and should not be limited to "only one" unless explicitly indicated to the contrary. Thus, for example, reference to "a component" includes embodiments having two or more such components unless the context clearly indicates otherwise.

When such a range is expressed, embodiments include from the one particular value and/or to the other particular value.

Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that any particular order be inferred.

Claim 1:
A cell culture vessel (<NUM>) comprising:
a top (<NUM>), a bottom (<NUM>), sidewalls (<NUM>. <NUM>) and a necked opening (<NUM>);
a substrate (<NUM>) on the bottom (<NUM>) of the vessel (<NUM>) comprising a plurality of microcavities (<NUM>), each microcavity (120a, 120b, 120c) of the plurality of microcavities (<NUM>) comprising a well opening (123a, 123b, 123c) and a concave well bottom (121a, 121b, 121c); and
an angled flange (<NUM>) surrounding at least a portion (<NUM>) of the substrate (<NUM>) to define a perimeter (<NUM>) at a top of the flange (<NUM>),
wherein the flange (<NUM>) has an inner face adjacent to the portion (<NUM>) of the substrate (<NUM>) and an outer face (<NUM>) opposite the portion (<NUM>) of the substrate (<NUM>); and
wherein the outer face (<NUM>) of the flange (<NUM>) is angled from the perimeter (<NUM>) to a base (<NUM>) of a channel (<NUM>).