Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
  • C12M23/08—Flask, bottle or test tube
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
  • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/38—Caps; Covers; Plugs; Pouring means
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0848—Specific forms of parts of containers
  • B01L2300/0858—Side walls

Definitions

• the present disclosure relates generally to cell culture vessels and methods of culturing cells, and more particularly, to cell culture vessels for containing three-dimensional cells and methods of culturing three-dimensional cells in the cell culture vessel.
• a cell culture vessel has sidewalls and a bottom surface.
• the bottom surface is a cell culture surface having a plurality of microcavities.
• the cell culture surface is a substrate attached to the sidewalls.
• the sidewalls are attached to the substrate so that there are no flat surfaces around the periphery of the cell culture surface.
• the vessel can include a top, a bottom, sidewalls, a necked opening or aperture and an endwall, opposite the necked opening.
• the cell culture vessel may have a lid.
• the cell culture vessel may have a lid, which may be the top of the vessel, and a necked opening or aperture.
• the interior surface of the bottom of the vessel is, in embodiments, the cell culture surface.
• the cell culture surface can span a length of the cell culture chamber.
• the vessel can include a neck portion of the inner surface of the wall extending from the aperture to the cell culture surface at an angle.
• a method of culturing cells in the cell culture vessel can include passing liquid through the aperture from outside the vessel into the cell culture chamber, thereby providing a predetermined amount of liquid to the cell culture chamber.
• each microcavity of the plurality of microcavities has a concave bottom surface and an opening at the top. Liquid enters each microcavity through the opening at the top of each microcavity.
• the vessel can have a necked opening or aperture that can be closed with a cap.
• the top wall of the vessel may be a lid.
• the lid can be opened, with a sliding opening or with a hinged opening, or with any other known opening mechanism.
• the vessel does not have a necked opening, and has a lid instead.
• a method of culturing cells in the cell culture vessel can include introducing a predetermined amount of liquid, such as liquid media, containing cells, into the cell culture chamber, and depositing at least a portion of the predetermined amount of liquid in at least one microcavity of the plurality of microcavities.
• the method can further include culturing cells in the at least one microcavity of the plurality of microcavities after depositing the at least a portion of the predetermined amount of liquid in the at least one microcavity.
• FIG. 1 schematically illustrates a side view of a cell culture vessel in accordance with embodiments of the disclosure
• FIG. 2 shows a plan view of cell culture vessel along line 2-2 of FIG. 1 in accordance with embodiments of the disclosure
• FIG. 3 shows a cross-sectional view of cell culture vessel along line 3-3 of FIG. 1 in accordance with embodiments of the disclosure
• FIG. 4 shows a cross-sectional view of the cell culture vessel along line 4-4 of FIG. 1 in accordance with embodiments of the disclosure
• FIG. 5 illustrates an enlarged schematic representation of a cell culture vessel taken at view 5 of FIG. 4 including a cell culture surface having a plurality of microcavities in accordance with embodiments of the disclosure
• FIG. 6 shows a cross-sectional view of a cell culture vessel having a cell culture surface having a plurality of microcavities along line 6-6 of FIG. 5 in accordance with embodiments of the disclosure
• FIG. 7 shows an alternative embodiment of the cross-sectional view of a cell culture vessel including a cell culture surface having a plurality of microcavities of FIG. 6 in accordance with embodiments of the disclosure
• FIG. 8 shows a partial cross-sectional view of a cell culture vessel along line
• FIG. 8- 8 of FIG. 2 including a lid portion and a groove in accordance with embodiments of the disclosure
• FIG. 9 shows a partial cross-sectional view of a cell culture vessel along line
• FIG. 9- 9 of FIG. 2 including a lid and a hinge in accordance with embodiments of the disclosure
• FIG. 10 schematically illustrates a side view of three cell culture vessels that are stacked together in accordance with embodiments of the disclosure
• FIG. 11 is a cross-sectional view of an alternative embodiment of the cell culture vessel of FIG. 3 including a method of culturing cells in the fifth exemplary cell culture vessel in accordance with embodiments of the disclosure;
• FIG. 12 shows a step of the method of culturing cells in the cell culture vessel of FIG. 11 in accordance with embodiments of the disclosure
• FIG. 13 is an enlarged schematic representation of a cell culture vessel taken at view 13 of FIG. 12 including a cell culture surface having a plurality of microcavities in accordance with embodiments of the disclosure;
• FIG. 14 shows a step of the method of culturing cells in the cell culture vessel of FIG. 11 in accordance with embodiments of the disclosure
• FIG. 15 is an enlarged schematic representation of a cell culture vessel taken at view 15 of FIG. 14 including a cell culture surface having a plurality of microcavities and a method of culturing cells in at least one microcavity of the plurality of microcavities in accordance with embodiments of the disclosure;
• FIG. 16 shows a step of the method of culturing cells in the cell culture vessel of FIG. 4 in accordance with embodiments of the disclosure.
• FIG. 17 illustrates a side view of a cell culture vessel in accordance with embodiments of the disclosure
• FIG. 18 is a plan view of a cell culture vessel along line 18-18 of FIG. 17 in accordance with embodiments of the disclosure.
• FIG. 19 is a cross-sectional view of an embodiment of a cell culture vessel along line 19-19 of FIG. 18 including a recess in accordance with embodiments of the disclosure;
• FIG. 20 is a cross-sectional view of an alternative embodiment of the cell culture vessel of FIG. 29 including a recess in accordance with embodiments of the disclosure;
• FIG. 21 is an enlarged schematic illustration of a portion of the third exemplary cell culture vessel taken at view 21 of FIG. 19 including a cell culture surface including a plurality of microcavities positioned in a recess in accordance with embodiments of the disclosure;
• FIG. 22 shows an alternative embodiment of the portion of the cell culture vessel having a cell culture surface having a plurality of microcavities positioned in a recess of FIG. 21 including a stepped portion in accordance with embodiments of the disclosure;
• FIG. 23 shows an alternative embodiment of the cell culture vessel having a cell culture surface including a plurality of microcavities positioned in a recess of FIG. 21 including a peripheral surface of the cell culture surface in accordance with embodiments of the disclosure;
• FIG. 24 shows a cross-sectional view of an exemplary embodiment of the third exemplary cell culture vessel along line 24-24 of FIG. 17 including a recess in accordance with embodiments of the disclosure
• FIG. 25 shows a cross-sectional view of an exemplary cell culture vessel of FIG. 19 including a protrusion in accordance with embodiments of the disclosure
• FIG. 26 is a cross-sectional view of a cell culture vessel including a protrusion of FIG. 25 in accordance with embodiments of the disclosure.
• FIG. 27 is an enlarged schematic representation of an embodiment of a portion of the cell culture vessel taken at view 28 of FIG. 25 including a cell culture surface including a plurality of microcavities positioned on a protrusion in accordance with embodiments of the disclosure;
• FIG. 28 shows an embodiment of the portion of the cell culture vessel including a cell culture surface including a plurality of microcavities positioned on a protrusion of FIG. 27 including a stepped portion in accordance with embodiments of the disclosure;
• FIG. 29 is a cross-sectional view of a cell culture vessel of FIG. 24 including a protrusion in accordance with embodiments of the disclosure
• FIG. 30 is a cross-sectional view of a cell culture vessel of FIG. 19 including a predetermined amount of liquid in accordance with embodiments of the disclosure
• FIG. 31 is a cross-sectional view of a cell culture vessel including a predetermined amount of liquid of FIG. 30 in accordance with embodiments of the disclosure
• FIG. 32 is an enlarged schematic representation of an embodiment of a portion of the cell culture vessel taken at view 32 of FIG. 30 including submerged surfaces of the vessel in accordance with embodiments of the disclosure;
• FIG. 33 shows an alternative embodiment of the portion of the cell culture vessel including submerged surfaces of the vessel of FIG. 32 including a recess in accordance with embodiments of the disclosure;
• FIG. 34 shows an alternative exemplary embodiment of the portion of a cell culture vessel including submerged surfaces of the vessel of FIG. 32 including a protrusion in accordance with embodiments of the disclosure
• FIG. 35A and FIG. 35B are drawings illustrating cells growing as spheroids in microcavities and cells growing in irregular shapes on flat surfaces in a cell culture vessel.
• FIG. 36A is a photograph of spheroids in an array of microcavities.
• FIG. 36B is a photograph of cells growing in irregular cellular conglomerates.
• a cell culture vessel e.g., flask
• 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.
• 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.
• 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.
• Cell culture vessels can be structured and arranged to provide an appropriate environment for cells to form spheroids in culture.
• the cell culture vessel can include a cell culture surface including a plurality of microcavities (e.g., microcavities, micron-sized wells, submillimeter-sized wells).
• microcavities e.g., microcavities, micron-sized wells, submillimeter-sized wells.
• the disclosure provides cell culture vessels that do not have flat surfaces that cells can settle on and grow in an irregular multicellular form. That is, the cell culture surface of the vessel consists substantially of microcavities.
• the cell culture surface can be an insert placed in the flask or the cell culture surface can be bonded to the wall of the cell culture vessel.
• a cell culture surface having an array of microcavities can be bonded to the wall of a cell culture vessel by, for example gluing, laser welding, ultrasonic welding, or some other method.
• the cell culture surface can include top and/or bottom sides that include undulating (e.g., sinusoidal) surfaces that form the plurality of microcavities.
• the vessel 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).
• a material e.g., media, solid, liquid, gas
• three-dimensional cell cultures e.g., cell aggregates, spheroids
• 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.
• 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 or bottom defining a well
• 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.
• the spheroids can consume media (e.g., food, nutrients) and produce metabolite (e.g., waste) as a byproduct.
• media e.g., food, nutrients
• metabolite e.g., waste
• 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.
• FIG. 1 illustrates a side view of an embodiment of a cell culture vessel 100
• FIG. 2 shows a plan view of the vessel 100 along line 2-2 of FIG. 1.
• the cell culture vessel 100 can include a top 101, a bottom 108, a necked opening 112 and a port, shown in FIG. 1 covered by a cap 104.
• FIG. 2 a plan view, shows wall 107 surrounding the cell culture surface 115.
• FIG. 2 shows a plan view of these that features, top 101 bottom 108, and wall 107 (shown in FIG. 2) and the necked opening 112 have interior surfaces.
• top 101 has an interior surface 201
• wall 107 has an interior surface 207
• bottom 108 has an interior surface 208
• necked opening 112 has an interior surface 212.
• These interior surfaces define the cell culture chamber 103.
• the interior surface 208 of the bottom 108 of the vessel 100 is the cell culture surface 115.
• This cell culture surface 115 has an array of microcavities (See FIGS. 5-7) for containing and culturing spheroids.
• the interior surface of the wall abuts the cell culture surface 115.
• the cell culture surface 115 is made up of microcavities entirely.
• the cell culture surface consists essentially of microcavities.
• the vessel can be manufactured from a material including, but not limited to, polymer, polycarbonate, glass, and plastic.
• the vessel 100 is illustrated as being manufactured from a clear (e.g., transparent) material; although, in some embodiments, the vessel 100 may, alternatively, be manufactured from a semi-transparent, semi-opaque, or opaque material without departing from the scope of the disclosure.
• FIG. 3 shows a cross-sectional view of the vessel 100 along line 3-3 of FIG. 1.
• the cell culture surface 115 and the inner surface 207 of the wall 107 defines a cell culture chamber 103 of the vessel 100, with an aperture 105 extending through the wall 107 in fluid communication with the cell culture chamber 103.
• the cell culture chamber 103 can include an internal spatial volume of the vessel 100.
• the vessel 100 can include a cap 104 oriented to cover the aperture 105 to at least one of seal and block the aperture 105, thereby obstructing a path into the cell culture chamber 103 from outside the vessel 100 through the aperture 105.
• the cap 104 is removed and, therefore, not shown in other drawing figures, although it is to be understood that the cap 104 can be provided and selectively added to or removed from the aperture 105 of the vessel 100, in some embodiments, without departing from the scope of the disclosure.
• the cap 104 can include a filter that permits the transfer of gas in to and/or out of the cell culture chamber 103 of the vessel 100.
• the cap 104 can include a gas-permeable filter oriented to regulate a pressure of gas within the cell culture chamber 103, thereby preventing pressurization (e.g., over-pressurization) of the cell culture chamber 103 relative to a pressure of the environment (e.g., atmosphere) outside the vessel 100.
• pressurization e.g., over-pressurization
• FIG. 3 which shows a cross-sectional view along line 3-3 of
• FIG. 5 shows an enlarged schematic representation of a portion of the cell culture surface 115 taken at view 5 of FIG. 4.
• FIG. 6 shows a cross-sectional view of the portion of the cell culture surface 115 along line 6-6 of FIG. 5, and
• FIG. 7 shows an alternative embodiment of the cross-sectional view of FIG. 6.
• microcavities 120 can be arranged in a diagonal array, although other arrangements can be provided in other embodiments.
• each microcavity 120a, 120b, 120c can include a concave bottom 121a,
• each microcavity 120a, 120b, 120c can include an opening 123a, 123b, 123c in the top of each microcavity 120.
• the first side 125 of the cell culture surface 115 can include a non-linear (e.g., undulating, sinusoidal) profile and a second side 126 of the cell culture surface 115 can be flat.
• both the first side 125 and the second side 126 of the cell culture surface 115 can include a non-planar (e.g., undulating, sinusoidal) profile.
• the cell culture surface 115, and the vessel 100 (as discussed in FIG. 1-16) and 300 (as discussed in FIG. 17-34) 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.
• a polymeric material including, but not limited to, polystyrene, polymethylmethacrylate, polyvinyl chloride, polycarbonate, polysulfone, polystyrene copolymers, fluoropolymers, polyesters, polyamide
• At least a portion of the well 122a, 122b, 122c defined by the concave bottom 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.
• 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.
• each microcavity 120a, 120b, 120c of the plurality of microcavities 120 (as discussed in FIG. 1-16) and 320 (as discussed in FIG. 17-
• the plurality of microcavities can include a variety of features and variations of those features without departing from the scope of the disclosure.
• the plurality of microcavities can include a variety of features and variations of those features without departing from the scope of the disclosure.
• the plurality of microcavities can include a variety of features and variations of those features without departing from the scope of the disclosure.
• the plurality of microcavities can include a variety of features and variations of those features without departing from the scope of the disclosure.
• ⁇ 120 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.
• the opening 123a, 123b, 123c can include a variety of shapes.
• 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.
• the opening 123a, 123b, 123c can include a dimension
• the opening 123a, 123b, 123c can include a dimension of 100 ⁇ , 150 ⁇ , 200 ⁇ , 250 ⁇ m, 300 ⁇ , 350 ⁇ m, 400 ⁇ , 450 ⁇ , 500 ⁇ , 550 ⁇ , 600 ⁇ , 650 ⁇ , 700 ⁇ , 750 ⁇ , 800 ⁇ , 850 ⁇ , 900 ⁇ , 950 ⁇ , 1000 ⁇ , 1500 ⁇ , 2000 ⁇ , 2500 ⁇ , 3000 ⁇ , 3500 ⁇ , 4000 ⁇ , 4500 ⁇ , 5000 ⁇ , and any dimension or ranges of dimensions encompassed within the range of from about 100 ⁇ to about 5000 ⁇ .
• the well 122a, 122b, 122c (as discussed in FIG. 1-16) and 322 (as discussed in FIG. 17-34) defined by the concave surface 121a, 121b, 121c can include a variety of shapes.
• 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.
• 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 100 microns ( ⁇ ) to about 5000 ⁇ .
• the depth of the well 122a, 122b, 122c can include a dimension of 100 ⁇ , 150 ⁇ , 200 ⁇ , 250 ⁇ , 300 ⁇ , 350 ⁇ , 400 ⁇ , 450 ⁇ , 500 ⁇ , 550 ⁇ , 600 ⁇ , 650 ⁇ , 700 ⁇ , 750 ⁇ , 800 ⁇ , 850 ⁇ , 900 ⁇ , 950 ⁇ , 1000 ⁇ , 1500 ⁇ , 2000 ⁇ , 2500 ⁇ , 3000 ⁇ , 3500 ⁇ , 4000 ⁇ , 4500 ⁇ , 5000 ⁇ , any dimension or ranges of dimensions encompassed within the range of from about 100 ⁇ to about 5000 ⁇ .
• three-dimensional cells 150 e.g., spheroids, organoids 150a, 150b, 150c
• a dimension e.g., diameter
• the vessel 100 can include a neck portion 555 extending from the aperture 105 to the cell culture surface 115.
• the neck portion 555 can include one or more of an inclined (e.g., canted) profile, a profile that narrows in a direction toward and/or away from the aperture 105, and a profile that widens in a direction toward and/or away from the cell culture surface 115. As shown in FIG. 3, in some embodiments, the neck portion 555 is angled with respect to the cell culture chamber 103. Additionally, in some embodiments, the neck portion 555 of the vessel can have a bend 158. In embodiments, the bend 158 is higher than the cell culture surface 115. However, it is to be understood that, in some embodiments, the bend can be of any shape. For example, the bend 158 can be curved or sloped or angled or stepped.
• the cell culture vessel 100 can include a dam 130 extending from interior surface 212 of the neck portion 112.
• the dam 130 can include a port- facing surface 131 obstructing a path defined between the aperture 105 and the cell culture surface 115.
• the port-facing surface 131 of the dam 130 can be substantially perpendicular to the axis 510 of the vessel 100.
• at least a portion of a free end 135 of the dam 130 can be spaced a distance "dlO" from the inner surface 201 of the top 101.
• access to a rear portion of the vessel 100 can be provided.
• one or more instruments can be inserted into the aperture 105 of the vessel 100 past the dam 130 (e.g., through the distance "dlO") to access a region of the cell culture chamber 103 positioned behind the dam 130.
• the dam 130 of the cell culture vessel 100 can, likewise, at least one of obstruct and slow a velocity of material (e.g., food, nutrients) flowing in to and/or material (e.g., waste) flowing out of the vessel 100 while also permitting bulk access into the cell culture chamber 103 of the vessel 100.
• material e.g., food, nutrients
• material e.g., waste
• the vessel 100 can include a lid
• the lid 137 when closed, can be the top 101 of the vessel 100.
• the first major surface 538 can define at least a portion of the cell culture chamber 103.
• the lid 137 can be open or closed, or partially open (or partially closed). The lid 137 can be slidingly atached to the vessel 100 or, in embodiments, the lid 137 can be hingedly attached to the vessel 100.
• the aperture 536 can be in fluid communication with the cell culture chamber 103.
• a first distance “dll” from the cell culture surface 115 to top 101 or lid 137 can be less than a second distance “dl2" from the cell culture surface 115 to the opening 507 of the aperture 105.
• the second distance "dl2" from the cell culture surface 115 to the opening 507 of the aperture 105 can be defined at any location of the opening 507.
• the second distance "dl2" from cell culture surface 115 to the opening 507 of the aperture 105 can be defined as the closest location of the opening 507 of the aperture 105 relative to the cell culture surface 115.
• the second distance "dl2" from the cell culture surface 115 to the opening 507 of the aperture 105 can be defined as the lowermost location of the opening 507 of the aperture 105 relative to the direction of gravity "g".
• one or more features of the vessel 100 can provide a cell culture chamber 103 including a larger volume in which material can be contained.
• the second distance "dl2" from the cell culture surface 115 to the opening 507 of the aperture 105 can define a maximum fill line with respect to a volume of material that can be contained within the cell culture chamber 103 of the vessel 100.
• the maximum fill line of a volume of material contained within the cell culture chamber 103 would be commensurate with the distance from the lid 137 as any additional material added to the cell culture chamber 103 would flow out of the opening of the aperture 105 rather than being contained within the cell culture chamber 103.
• the vessel 100 can include a volume including a portion of the cell culture chamber 103 that is not employed with respect to containing material. Accordingly, in some embodiments, by providing the vessel 100, in accordance with embodiments of the disclosure, including the second distance "dl2" that is greater than the first distance "dll", the entire volume of the cell culture chamber 103 can be employed to contain material, a larger volume of material can be contained in the cell culture chamber 103, and an efficient allocation of material and overall utilization of space of the vessel 100 can be achieved. Likewise, in some embodiments, the second distance "dl2" can be equal to the first distance "dll", without departing from the scope of the disclosure.
• 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.
• media e.g., food, nutrients
• media e.g., waste
• 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.
• 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.
• one or more features of the cell culture vessel 100 and the methods of culturing cells 150 in the cell culture vessel 100 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 103 of the vessel 100, added to the cell culture chamber 103, and removed from the cell culture chamber 103, thereby providing a desirable, effective environment in which to culture three-dimensional cells.
• FIG. 8 which shows a partial cross-sectional view of the vessel
• top wall 101 can include a groove 180, and the lid 137 can be slideable within the groove 180 (as shown by arrow 181 in FIG. 2) to selectively provide access to the cell culture chamber 103, for example, through an opening of aperture 136.
• the vessel 100 can include a hinge 182 connecting the lid 137 to the wall 101.
• the lid 137 can be rotatable about the hinge 182 (as shown by arrow 183) to selectively provide access to the cell culture chamber 103, for example, through an opening of aperture 136.
• the lid 137 can be connected to the wall 101 with one or more fasteners (not shown) and/or adhesives (not shown) including reusable and non- reusable adhesives to, for example, selectively provide access to the cell culture chamber 103, for example, through an opening of aperture 136.
• a length "L2" of the vessel 100 measured from the port 105 to the end wall 107 extending along the axis 510 of the vessel can be equal to or greater than the length "LI" of the cell culture surface 115.
• a plurality of vessels 100 can be stacked (e.g., vertically relative to the direction of gravity) to, for example, reduce a surface area (e.g., laboratory surface area, table surface area) occupied by the plurality of vessels 100.
• FIG. 10 schematically illustrates a side view of a plurality of vessels 100, 100a, 100b stacked together in accordance with embodiments of the disclosure.
• vessel 100a including wall 101a, cap 104a, neck portion 555a, lid 137a, and a bottom 108a can be stacked, one on top of the other.
• the bottom portion can be stacked, one on top of the other.
• the bottom portion can be stacked, one on top of the other.
• the bottom portion 108 of vessel 100 can be positioned on (e.g., facing) the lid
• the plurality of vessels 100, 100a, 100b can efficiently utilize a space (e.g., area, volume) in which the vessels 100, 100a, 100b are provided, for example, during one or more of storage, cleaning, and culturing with respect to the vessels 100, 100a, 100b.
• a space e.g., area, volume
• the vessels 100, 100a, 100b are provided, for example, during one or more of storage, cleaning, and culturing with respect to the vessels 100, 100a, 100b.
• a space e.g., area, volume
• two vessels or more than three vessels can be stacked in accordance with embodiments of the disclosure, without departing from the scope of the disclosure.
• the plurality of vessels 100, 100a, 100b can be positioned separately and/or together (e.g., stacked) in a variety of configurations including configurations not explicitly disclosed in the disclosure, without departing from the scope of the disclosure.
• the vessels are stackable. Moreover, based at least in part on one or more features of the bend 158 in the neck 112, when stacked in accordance with embodiments of the disclosure, access to the ports 105, 105a, 105b can be maintained to, for example, permit addition of material (e.g., food, nutrients) and/or removal of material (e.g., waste) from the respective cell culture chambers 103, 103a, 103b while the plurality of vessels 100, 100a, 100b are stacked (e.g., stationary). For example, in some embodiments, stacking vessels that do not include one or more features of the disclosure could one or more of limit, obstruct, and prevent access to the openings of the apertures.
• material e.g., food, nutrients
• material e.g., waste
• stacked vessels including openings of the apertures to which access is one or more of limited, obstructed, and prevented may be moved (e.g., at least one of translated and rotated) relative to each other during a culturing process to, for example, provide access to the openings of the apertures.
• movement of vessels relative to each other during a culturing process may one or more of dislodge and disturb cells being cultured in the vessels, thereby negatively impacting the cell culture process.
• a method of culturing cells 150 (See FIG. 15) in the cell culture vessel 100 can include passing liquid (e.g., represented by arrow 106) through the aperture 105 from outside the vessel 100 into the cell culture chamber 103, thereby providing a predetermined amount of liquid 140 in the cell culture chamber 103.
• the method can be performed in a vessel with or without the optional dam 130.
• the method can include tilting the vessel 100 so that the bend 158 forms a low point in the neck 112.
• Liquid 140 introduced into the vessel can accumulate in the neck 112 at the bend 158. That is, the vessel can contain a predetermined amount of liquid 140 in the bend 158 of the neck 112, without liquid 140 contacting the microcavity array 115.
• preventing liquid 140 from contacting one or more microcavities 120 of the cell culture surface 115 containing an array of microcavities 120, at this stage of the method can provide several advantages that, for example, facilitate improved culturing of the cells 150 (See FIG. 15).
• the vessel can be tilted slowly back so that axis 510 is perpendicular to gravity "g", allowing the liquid 140 to slowly flow across the cell culture surface 115 having an array of microcavities 120, allowing the liquid to slowly fill the microcavities 120.
• This step is shown schematically in FIG. 12.
• FIG. 13 illustrates an enlarged schematic representation of a cell culture vessel 100 taken at view 13 of FIG. 12 showing at least a portion of the liquid 140 flowing from the neck 112 on to the cell culture surface 115 along the length "LI" of the cell culture chamber 103 and entering microcavities 120a, 120b, 120c of the microcavity array 120.
• the movement of the vessel 100 to cause the liquid to flow can be controlled and slow (e.g., performed during a duration of time on the order of minutes).
• microcavities 120a, 120b, 120c of the plurality of microcavities 120 can result in bubbles forming in the microcavities.
• This slow introduction of liquid to the microcavities 120 allows liquid to flow into the microcavities with reduced bubble formation. Bubbles disrupt cell growth.
• Liquid media can have a high surface tension (it can be thick) and the microcavities 120 are very small.
• bend 158 of the neck 112 can abut the cell culture surface 115 and fluid 140 can flow from the bend 158 in the neck 112 and deposit into at microcavities 120a, 120b, 120c with controlled flow (e.g., reduced or no liquid splashing and reduced or no turbulent flow), thereby providing a steady flow of liquid depositing into the well 122a, 122b, 122c of the microcavities 120a, 120b, 120c through a portion of the respective opening 123a, 123b, 123c of the microcavities 120a, 120b, 120c while displacing gas from the well 122a, 122b, 122c.
• controlled flow e.g., reduced or no liquid splashing and reduced or no turbulent flow
• the cell culture surface 115 extends from wall 107 to wall 107. In embodiments, the cell culture surface 115 does not have any flat areas. That is, the cell culture surface is an array of microcavities from wall to wall with no border, no flat areas between the cell culture surface and walls 107. In embodiments, the cell culture surface consisting essentially of a plurality of microcavities. In embodiments, there no flat areas in the cell culture chamber for cells to settle on. This is important to ensure that cells do not settle in the cell culture chamber outside of the microwells. When cells settle outside of microwells, on flat areas outside the cell culture surface, cells can grow as irregular cellular conglomerates, and create an inhomogeneous population of multicellular 3D structures in the vessel.
• a cell culture surface consisting essentially of a plurality of microcavities. When cells settle outside of microwells, on flat areas outside the cell culture surface, cells can grow as irregular cellular conglomerates 801 (See FIG. 35A and 35B, 36A and 36B), and create an inhomogeneous population of multicellular 3D structures in the vessel. In embodiments, a cell culture surface consisting essentially of a plurality of microcavities.
• the cell culture surface 115 extends from wall 107 to wall
• the cell culture surface 115 does not have any flat areas. That is, the cell culture surface is an array of microcavities 120 extending from wall to wall with no border, no flat areas between the cell culture surface and walls 107. In embodiments, the cell culture surface consisting essentially of a plurality of microcavities. In embodiments, there no flat areas in the cell culture chamber for cells to settle on. This is important to ensure that cells do not settle in the cell culture chamber outside of the microwells. When cells settle outside of microwells, on flat areas outside the cell culture surface, cells can grow as irregular cellular conglomerates 801 (See FIG. 35A and 35B, 36A and 36B), and create an inhomogeneous population of multicellular 3D structures in the vessel. In embodiments, a cell culture surface consisting essentially of a plurality of microcavities.
• the liquid 140 can be caused to flow from the neck 112 over the entire cell culture surface 115 based at least on the movement of the vessel 100.
• FIG. 15 illustrates an enlarged schematic representation of a cell culture vessel 100 taken at view 15 of FIG. 14 including a method of culturing cells 150 in the cell culture vessel 100.
• the method can include culturing cells 150 (e.g., spheroid 150a, spheroid 150b, spheroid 150c) in the at least one microcavity 120a, 120b, 120c of the plurality of microcavities 120 after depositing the at least a portion of the predetermined amount of liquid 140 in the at least one microcavity 120a, 120b, 120c.
• the axis 510 of the vessel 100 can be substantially perpendicular relative to the direction of gravity "g" while culturing cells 150 in the microcavities 120.
• the method can further include adding additional liquid media 140 (as shown by arrow 508) to the cell culture chamber 103.
• additional liquid media 140 cell food, liquid containing nutrients
• the vessel can be filled right up to the lid 137 or top 101 of the vessel 100. In this way, the cell culture chamber 103 can contain larger volume of liquid 140 than it would be able to contain if the port were arranged lower with respect to the top 101 of the vessel 100.
• FIGS. 17-34 show an embodiments without a necked opening or a port, it is to be understood that the embodiments shown in FIGS. 17-34 can be incorporated into the embodiments illustrated in FIGs. 1-16, a vessel with a port.
• FIG. 17 schematically illustrates a side view of cell culture vessel 300
• FIG. 18 schematically illustrates a plan view of the vessel 300 along line 18-18 of FIG. 17.
• the cell culture vessel 300 can include a wall 301 and a lid 304.
• the vessel 300 is illustrated as being manufactured from a clear (e.g., transparent) material; although, in some embodiments, the vessel 300 can, alternatively, be manufactured from a semi-transparent, semi-opaque, or opaque material without departing from the scope of the disclosure.
• the lid 304 can be oriented to cover an opening of the vessel 300 to at least one of seal and block the opening, thereby obstructing a path into the cell culture chamber 303 from outside the vessel 300 through the opening.
• the lid 304 is removed and, therefore, not shown in other drawing figures, although it is to be understood that the lid 304 can be provided and selectively added to or removed from the opening of the vessel 300, in some embodiments, without departing from the scope of the disclosure.
• the lid 304 can include a filter that permits the transfer of gas in to and/or out of a cell culture chamber 303 (See FIG. 19) of the vessel 300.
• the lid 304 can include a gas-permeable filter oriented to regulate a pressure of gas within the cell culture chamber 303, thereby preventing pressurization (e.g., over-pressurization) of the cell culture chamber 303 relative to a pressure of the environment (e.g., atmosphere) outside the vessel 300.
• pressurization e.g., over-pressurization
• FIG. 19 shows a cross-sectional view of an exemplary embodiment of the cell culture vessel 300 along line 19-19 of FIG. 18, and FIG. 20 shows an alternative exemplary embodiment of the cross-sectional view of the cell culture vessel 300 of FIG. 19.
• FIG. 25 shows an alternative exemplary embodiment of the cross-sectional view of the cell culture vessel 300 of FIG. 18, and FIG. 26 shows an alternative exemplary embodiment of the cross-sectional view of the cell culture vessel 300 of FIG. 25.
• the wall 301 can include an inner surface 302, and the vessel 300 can include a cell culture surface 315 including a plurality of microcavities 320. As shown in FIG. 19 and FIG.
• the cell culture surface 315 and the inner surface 302 of the wall 301 define the cell culture chamber 303 of the vessel 300.
• the inner surface 302 of the wall 301 can define a cell culture chamber 303 of the vessel 300 including, for example, a first region 303a and a second region 303b, and the cell culture surface 315 can be positioned in the cell culture chamber 303, between the first region 303a and the second region 303b.
• an outer perimeter As shown in FIG. 19 and FIG. 20, in some embodiments, an outer perimeter
• FIG. 24 shows an exemplary cross-section view of the vessel 300 taken along line 24-24 of FIG. 30, where the entire outer perimeter 330 laterally circumscribes the plurality of microcavities 320 and the entire outer perimeter 330 is positioned in the recess 335.
• the outer perimeter 330 of the cell culture surface 315 can surrounds the plurality of microcavities 320, and at least a portion 331 of the outer perimeter 330 can be positioned on a protrusion 337 of the inner surface 302 of the wall 301 of the vessel 300.
• FIG. 29 shows an alternative exemplary embodiment of the cross- sectional view of the third exemplary cell culture vessel 300 of FIG. 24, where the entire outer perimeter 330 laterally circumscribes the plurality of microcavities 320 and the entire outer perimeter 330 is positioned on the recess 335 protrusion 337.
• “surrounds” means that, in a top or bottom view in a direction perpendicular to a major feature of the cell culture surface 315, for example, an outer periphery defined by the first feature surrounds an outer periphery defined by the second feature.
• an outer periphery (defined by the outer perimeter 330) of the cell culture surface 315 surrounds an outer periphery (defined by the plurality of microcavities 320) of the cell culture surface 315.
• the cell culture surface 315 extends from wall 301 to wall 301. In embodiments, the cell culture surface 315 does not have any flat areas. That is, the cell culture surface is an array of microcavities 320 extending from wall to wall with no border, no flat areas between the cell culture surface and walls 301. In embodiments, the cell culture surface consisting essentially of a plurality of microcavities. In embodiments, there no flat areas in the cell culture chamber for cells to settle on. This is important to ensure that cells do not settle in the cell culture chamber outside of the microwells. When cells settle outside of microwells, on flat areas outside the cell culture surface, cells can grow as irregular cellular conglomerates 801 (See FIG. 35A and 35B, 36A and 36B), and create an inhomogeneous population of multicellular 3D structures in the vessel. In embodiments, a cell culture surface consisting essentially of a plurality of microcavities.
• FIGS. 21-23 show exemplary embodiments of an enlarged view of a portion of the vessel 300 taken at view 21 of FIG. 19 including the at least a portion 331 of the outer perimeter 330 of the cell culture surface 315 positioned in the recess 335.
• FIG. 27 and FIG. 28 show exemplary embodiments of an enlarged view of the vessel 300 taken at view 28 of FIG. 25 including the at least a portion 331 of the outer perimeter 330 positioned on the protrusion 337.
• each microcavity 320a, 320b, 320c of the plurality of microcavities 320 can include a concave surface 321a, 321b, 321c defining a well 322a, 322b, 322c and an opening 323a, 323b, 323c Liquid enters and exits the microcavities through the openings 323a, 323b, 323c.
• the cell culture surface 315 can be attached to the wall 301 of the vessel 300.
• the cell culture surface 315 can be attached to the wall 301 of the vessel 300 with an adhesive (not shown), a solvent (not shown), or a fastener (not shown), by welding (laser welding or ultrasonic welding) or the wall and the cell culture surface 315 can be molded together.
• the cell culture surface 315 can be attached to the inner surface 302 of the wall 301 of the vessel 300 based at least in part on, for example, operation of a plastic welding process, a laser welding process, an ultrasonic welding process.
• at least one of the wall 301 and the outer perimeter 330 can include an energy director (not shown) to facilitate bonding of the cell culture surface 315 to the wall 301 of the vessel 300 based at least in part on operation of the plastic welding process.
• the vessel 300 can include a stepped portion 306 extending outward from the cell culture surface 315 and forming the recess 335.
• the stepped portion 306 can increase at least one of a volume of the cell culture chamber 303 and a quantity of microcavities 320a, 320b, 320c of the plurality of microcavities 320 within the cell culture chamber 303.
• the stepped portion 306 can provide a relatively larger recess 335 without increasing a thickness of the wall 301 of the vessel 300 as compared to the corresponding recess 335 and wall 301 thickness shown, for example, in FIG. 21.
• the recess 335 formed by the stepped portion 306 can be oriented to accommodate the at least a portion 331 of the outer perimeter 330 of the cell culture surface 315 positioned in the recess 335.
• the vessel 300 can include a stepped portion 308 extending inward toward the cell culture surface 315 and forming the protrusion 337.
• the stepped portion 308 can provide a relatively larger protrusion 337 without increasing a thickness of the wall 301 of the vessel 300 as compared to the corresponding protrusion 337 and wall 301 thickness shown, for example, in FIG. 28.
• the protrusion 337 formed by the stepped portion 308 can be oriented to accommodate the at least a portion 331 of the outer perimeter 330 of the cell culture surface 315 positioned on the protrusion 337.
• the cell culture surface 315 extends from wall 301 to wall 301. In embodiments, the cell culture surface 315 does not have any flat areas. That is, the cell culture surface is an array of microcavities 320 extending from wall to wall with no border, no flat areas between the cell culture surface and walls 301. In embodiments, the cell culture surface consisting essentially of a plurality of microcavities. In embodiments, there no flat areas in the cell culture chamber for cells to settle on. This is important to ensure that cells do not settle in the cell culture chamber outside of the microwells. When cells settle outside of microwells, on flat areas outside the cell culture surface, cells can grow as irregular cellular conglomerates 801 (See FIG. 35A and 35B, 36A and 36B), and create an inhomogeneous population of multicellular 3D structures in the vessel. In embodiments, a cell culture surface consisting essentially of a plurality of microcavities.
• the at least a portion 331 of the outer perimeter 330 of the cell culture surface 315 can be spaced from the portion of the cell culture surface 315 including the openings 323a, 323b, 323c of the microcavities 320a, 320b, 320c in a direction away from the concave surface 321a, 321b, 321c of each microcavity 320a, 320b, 320c of the plurality of microcavities 320.
• the cell culture surface 315 can include a peripheral surface 332 extending from the at least a portion 331 of the outer perimeter 330 to the portion of the cell culture surface 315 including the openings 323a, 323b, 323c.
• the peripheral surface 332 can include a vertical orientation (e.g., extending in the direction of gravity); however, in some embodiments, the peripheral surface 332 can be inclined relative to the direction of gravity to, for example, direct cells toward the openings 323a, 323b, 323c of the microcavities 320a, 320b, 320c.
• the opening 323a of the microcavity 320a can be positioned to abut the inner surface 302 of the wall
• the opening 323a of the microcavity 320a can be flush with the inner surface 302 of the wall 301 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 surface 302 into the well 322a of the microcavity 320a without settling on or adhering to a surface of the vessel 300.
• the opening 323a of the microcavity 320a can be positioned to abut the peripheral surface 332 of the cell culture surface 315 with the outer perimeter 330 supported by the protrusion 337.
• the opening 323a of the microcavity 320a can be flush with the peripheral surface 332 of the cell culture surface
• the cell culture surface 315 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 peripheral surface 332 into the well 322a of the microcavity 320a without settling on or adhering to any other surface of the vessel 300.
• the cell culture surface 315 extends from wall 301 to wall 301.
• the cell culture surface 115 does not have any flat areas. That is, the cell culture surface is an array of microcavities 320 extending from wall to wall with no border, no flat areas between the cell culture surface and walls 301.
• the cell culture surface consisting essentially of a plurality of microcavities. In embodiments, there no flat areas in the cell culture chamber for cells to settle on. This is important to ensure that cells do not settle in the cell culture chamber outside of the microwells. When cells settle outside of microwells, on flat areas outside the cell culture surface, cells can grow as irregular cellular conglomerates 201 (See
• FIG. 35A and 35B, 36A and 36B create an inhomogeneous population of multicellular
• a cell culture surface consisting essentially of a plurality of microcavities.
• cells that settle on or adhere to a surface of the vessel are cells that settle on or adhere to a surface of the vessel.
• FIG. 35A and FIG. 35B are schematic drawings of cells accumulating in flat areas on the periphery of the microcavities. For example, in some embodiments, cells that do not fall (based at least on the force of gravity) into the well 322a,
• 322b, 322c and that accumulate or attach to other surfaces of the vessel 300 can grow outside of the well 322a, 322b, 322c.
• cells can grow as irregular cellular conglomerates 801 which are undesirable.
• irregular cellular conglomerates 801 can creep into neighboring microcavities and disrupt (e.g., discourage, alter, slow, or prevent) desired growth of three-dimensional cells within the well 322a, 322b, 322c.
• cells that accumulate or attach to other surfaces of the vessel 300 can grow and dislodge three-dimensional cells in the well 322a, 322b, 322c, thereby disrupting or destroying desired growth of three-dimensional cells within the well 322a, 322b, 322c and altering desired size uniformity of the cells.
• all cells suspended within the liquid can be directed into the wells 322a, 322b, 322c, thus reducing and eliminating problems that can otherwise occur if cells attach to surfaces of the vessel 300 outside the wells 322a, 322b, 322c.
• the cell culture vessel 300 and the cell culture surface 315 can be manufactured from the same material.
• the cell culture surface 315 including the plurality of microcavities 320 can be manufactured (e.g., formed, machined, pressed, extruded, molded, printed by operation of 3D printing, etc.) as an integral part of the wall 301 of the vessel 300, such that there is no distinct boundary between the cell culture surface 315 and the wall 301 of the vessel 300. As shown in FIG.
• the cell culture surface 315 and the inner surface 302 of the wall 301 can define the cell culture chamber 303 of the vessel 300.
• the inner surface 302 of the wall 301 can define the cell culture chamber 303 of the vessel 300, including a first region 303a and a second region 303b, and the cell culture surface 315 (integrally formed with the wall 301) can be positioned in the cell culture chamber 303 between the first region 303a and the second region 303b.
• the vessel 300 including the wall 301 and cell culture surface 315 integrally manufactured together can include a material that is non-permeable.
• the wall 301 of the vessel 300 can be manufactured from a non-permeable material, and the cell culture surface 315 can be manufactured from one or more of a non-permeable material, a non-porous material, a gas permeable material or a porous material, integrally formed with the wall 301.
• the cell culture surface 315 and the wall 301 are manufactured as a single part, the cell culture surface 315 extends from wall 301 to wall 301.
• the cell culture surface 315 does not have any flat areas. That is, the cell culture surface is an array of microcavities 320 extending from wall to wall with no border, no flat areas between the cell culture surface and walls 301. In embodiments, the cell culture surface consisting essentially of a plurality of microcavities. In embodiments, there no flat areas in the cell culture chamber for cells to settle on. This is important to ensure that cells do not settle in the cell culture chamber outside of the microwells. When cells settle outside of microwells, on flat areas outside the cell culture surface, cells can grow as irregular cellular conglomerates 201 (See FIG. 35A and 35B, 36A and 36B), and create an inhomogeneous population of multicellular 3D structures in the vessel. In embodiments, a cell culture surface consisting essentially of a plurality of microcavities.
• the vessel 300 can include a predetermined amount of liquid 370, and a method of culturing cells in the cell culture vessel 300 can include depositing liquid 370 in at least one microcavity 320a, 320b, 320c of the plurality of microcavities 320 and culturing cells in the at least one microcavity 320a, 320b, 320c after depositing the liquid 370 in the at least one microcavity 320a, 320b, 320c.
• FIG. 45 which shows an enlarged view of the integrally formed cell culture surface 315 and wall 301 of the vessel 300 at view 45 of FIG. 43
• the predetermined amount of liquid 370 can contact submerged surfaces 325 of the vessel 300 and occupy a region of the cell culture chamber 303 of the vessel 300.
• FIG. 46 shows an alternate exemplary embodiment of FIG. 45, including features of the at least a portion 331 of the outer perimeter 330 of the cell culture surface 315 positioned in the recess 335 of the wall 301 of the vessel 300 with the predetermined amount of liquid contacting submerged surfaces 325 of the vessel 300, including the peripheral surface 332 of the cell culture surface 315, and occupying a region of the cell culture chamber 303 of the vessel 300.
• FIG. 34 shows an alternate exemplary embodiment of FIG. 32, including features of the at least a portion 331 of the outer perimeter 330 of the cell culture surface 315 positioned on the protrusion 337 of the wall 301 of the vessel 300 with the predetermined amount of liquid 370 contacting submerged surfaces 325 of the vessel 300, including the peripheral surface 332 of the cell culture surface 315, and occupying a region of the cell culture chamber 303 of the vessel 300.
• submerged surfaces 325 are shown with thicker line weights in FIGS. 32-34 with the understanding that, in some embodiments, submerged surfaces 325 can include surfaces of the vessel 300 that are in contact with the predetermined amount of liquid 370. In some embodiments, the submerged surfaces 325 can be defined relative to the direction of gravity "g" at or during a specified step of a method of culturing cells in the vessel 300.
• the predetermined amount of liquid 370 can define a liquid level 371, where the submerged surfaces 325 include surfaces of the vessel 300 in contact with the predetermined amount of liquid 370 that are, relative to the direction of gravity "g", positioned below the liquid level 371 and, therefore, submerged in the predetermined amount of liquid 370.
• the level 371 of the predetermined amount of liquid 370 can define a planar free surface of the predetermined amount of liquid 370 spaced a distance from a portion 375 of the cell culture surface 315.
• the portion 375 of the cell culture surface 315 can include the openings 323a, 323b, 323c and the planar free surface defined by the liquid level 371 of the predetermined amount of liquid 370 can be spaced a distance from the portion 375 in a direction away from the concave surface 321a, 321b, 321c of each microcavity 320a, 320b,
• cells that settle on or adhere to a surface of the vessel 300 can accumulate and grow (e.g., multiply) outside of the microcavities 320a, 320b, 320c causing problems with respect to desired growth of three-dimensional cells within the microcavities
• cells that do not fall (based at least on the force of gravity) into the well 322a, 322b, 322c and that accumulate or attach to other surfaces of the vessel 300 can grow outside of the well 322a, 322b, 322c and disrupt (e.g., discourage, alter, slow, or prevent) desired growth of three-dimensional cells within the well 322a, 322b, 322c.
• the submerged surfaces 325 were to include planar surface portions parallel to the planar free surface of the predetermined amount of liquid 370, cells could accumulate or attach to the planar surface portions and could grow and dislodge three-dimensional cells in the well 322a, 322b, 322c, thereby disrupting or destroying desired growth of three-dimensional cells within the well 322a, 322b, 322c.
• all cells suspended within the liquid 370 can be directed into the wells 322a, 322b, 322c, thus reducing and eliminating problems that can otherwise occur if cells attach to surfaces of the vessel 300 outside the wells 322a, 322b, 322c.
• At least a portion 331 of the outer perimeter 330 of the cell culture surface 315 can be spaced a distance "d4" from the portion 375 of the cell culture surface 315 in a direction away from the concave surface 321a, 321b, 321c of each microcavity 320a, 320b, 320c of the plurality of microcavities 320.
• the cell culture surface 315 can include the peripheral surface 332 extending from the at least a portion 331 of the outer perimeter 330 to the portion 375 of the cell culture surface 315.
• a depth "d5" of the predetermined amount of liquid 370 from the liquid level 371 defining the planar free surface to the portion 375 of the cell culture surface 315 along the direction can be less than the distance "d4".
• at least a portion 331 of the outer perimeter 330 of the cell culture surface 315 can be spaced a distance "d6" from the portion 375 of the cell culture surface 315 in a direction away from the concave surface 321a, 321b, 321c of each microcavity 320a, 320b, 320c of the plurality of microcavities 320.
• the cell culture surface 315 can include the peripheral surface 332 extending from the at least a portion 331 of the outer perimeter 330 to the portion 375 of the cell culture surface 315.
• a depth "d7" of the predetermined amount of liquid 370 from the liquid level 371 defining the planar free surface to the portion 375 of the cell culture surface 315 along the direction can be less than the distance "d6".
• a depth "d5" of the predetermined amount of liquid 370 from the liquid level 371 defining the planar free surface to the portion 375 of the cell culture surface 315 along the direction can be less than the distance "d4" (e.g., FIG. 33, including the recess 335) and a depth "d7" of the predetermined amount of liquid 370 from the liquid level 371 defining the planar free surface to the portion 375 of the cell culture surface 315 along the direction can be less than the distance "d6" (e.g., FIG.
• all cells suspended within the liquid 370 can be directed into the wells 322a, 322b, 322c, thus reducing and eliminating problems that can otherwise occur if cells attach to surfaces of the vessel 300 outside the wells 322a, 322b, 322c.
• a method of culturing cells See FIG.
• the 16) in the vessel 300 can include depositing a portion of the predetermined amount of liquid 370 in at least one microcavity 320a, 320b, 320c of the plurality of microcavities 320a, 320b, 320c; and culturing cells in the at least one microcavity 320a, 320b, 320c after depositing the portion of the predetermined amount of liquid 370 in the at least one microcavity 320a, 320b, 320c.
• a method of culturing cells in the cell culture vessel 300 can include filling a region of a cell culture chamber 303 of the vessel 300 with a predetermined amount of liquid 370.
• the cell culture chamber 303 can be defined at least in part by an inner surface 302 of the wall 301 of the vessel 300, and the method can include depositing a portion of the predetermined amount of liquid 370 in at least one microcavity 320a, 320b, 320c of the plurality of microcavities 320 of the cell culture surface 315.
• the cell culture surface 315 can define at least a portion 331 of the region, and each microcavity 320a, 320b, 320c of the plurality of microcavities 320 can include a concave surface 321a, 321b, 321c defining a well 322a, 322b, 322c and an opening 323a, 323b, 323c in a portion 375 of the cell culture surface 315 defining a path into the well 322a, 322b, 322c.
• the method can further include culturing cells in the at least one microcavity 320a, 320b, 320c after depositing the portion of the predetermined amount of liquid 370 in the at least one microcavity 320a, 320b, 320c, with the predetermined amount of liquid 370 contacting submerged surfaces 325 of the vessel 300.
• the submerged surfaces 325 do not include planar surface portions including a surface normal that is opposite the direction of gravity "g".
• a level 371 of the predetermined amount of liquid 370 can define a planar free surface of the predetermined amount of liquid 370 that is perpendicular relative to the direction of gravity "g".
• submerged surfaces 325 that do not include planar surface portions including a surface normal that is opposite the direction of gravity "g"
• cells suspended in the liquid 370 will fall (e.g., based at least on the force of gravity) and/or be directed by the submerged surfaces 325 into the wells 322a, 322b, 322c of the microcavities 320a, 320b, 320c because there are no submerged surfaces 325 on which the cells can settle or to which the cells can adhere.
• cells that settle on or adhere to a surface of the vessel 300 can accumulate and grow (e.g., multiply) outside of the microcavities 320a, 320b, 320c causing problems with respect to desired growth of three- dimensional cells within the microcavities 320a, 320b, 320c.
• cells that do not fall (based at least on the force of gravity) into the well 322a, 322b, 322c and that accumulate or attach to other surfaces of the vessel 300 can grow outside of the well 322a, 322b, 322c and disrupt (e.g., discourage, alter, slow, or prevent) desired growth of three-dimensional cells within the well 322a, 322b, 322c.
• the submerged surfaces 325 were to include planar surface portions including a surface normal that is opposite the direction of gravity "g"
• cells could accumulate or attach to the planar surface portions and could grow and dislodge three-dimensional cells in the well 322a, 322b, 322c, thereby disrupting or destroying desired growth of three-dimensional cells within the well 322a, 322b, 322c.
• all cells suspended within the liquid 370 can be directed into the wells 322a, 322b, 322c, thus reducing and eliminating problems that can otherwise occur if cells settle on or attach to surfaces of the vessel 300 outside the wells 322a, 322b, 322c.
• planar surface portion is intended to mean any planar surface portion including a planar dimension greater than about 5 microns.
• submerged surfaces 325 that do not include planar surface portions parallel to the planar free surface of the level 371 of the predetermined amount of liquid 370 can be defined as submerged surfaces 325 that do not include planar surface portions, including a planar dimension greater than about 5 microns, parallel to the planar free surface of the level 371 of the predetermined amount of liquid 370.
• submerged surfaces 325 that do not include planar surface portions including a surface normal that is opposite the direction of gravity "g" can be defined as submerged surfaces 325 that do not include planar surface portions, including a planar dimension greater than about 5 microns, including a surface normal that is opposite the direction of gravity "g".
• the submerged surfaces 325 can include a planar portion; however, if a planar dimension of the planar surface portion is, for example, less than or equal to 5 microns, in some embodiments, the planar surface portion is considered too small for cells to reasonably accumulate or attach. Accordingly, in some embodiments, by providing submerged surfaces 325 that do not include planar surface portions including a planar dimension greater than about 5 microns, all cells suspended within the liquid 370 can be directed into the wells 322a, 322b, 322c, thus reducing and eliminating problems that can otherwise occur if cells settle on or attach to surfaces of the vessel 300 outside the wells 322a, 322b, 322c. In some embodiments, however, the submerged surfaces 325 can be entirely free of planar surface portions, irrespective of a threshold dimension defining the planar surface portion.
• FIG. 35A and FIG. 35B are drawings illustrating cells growing as spheroids in microcavities and cells growing as irregular cellular conglomerates 201. These irregular cellular conglomerates can occur in the embodiments shown in FIG. 1-16 (cell culture surface 115) or in the embodiments shown in FIG. 17-34 (cell culture surface 315), where flat surfaces occur in a vessel. It is important to avoid flat surfaces in the cell culture chamber 103 or 303, to avoid these irregular cellular conglomerates.
• FIG. 36A is a photograph of spheroids in an array of microcavities under suitable conditions provide a homogeneous population of spheroids in the vessel.
• FIG 36B is a photograph of irregular cellular conglomerates 801 isolated from a vessel having flat surfaces in the cell growth chamber 103, 303.
• embodiments of a cell culture vessel are provided which do not have flat surfaces in the cell culture surface, or do not have flat surfaces in the submerged region of the cell culture surface. That is, in embodiments of a cell culture vessel, the cell culture surface consists essentially of an array of microcavities, and does not provide flat surfaces that can produce undesirable irregular cellular conglomerates 801.
• 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.
• material can include fluid material (e.g., liquid or gas).
• material can include a culture solution or media including a liquid including solid particles (e.g., cells) suspended in the liquid.
• 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 cell culture surface and the vessel, prepare the cell culture surface for cellular growth and other uses of liquid.
• liquid can include a culture solution or media including a liquid including solid particles (e.g., cells) suspended in the liquid.
• gas can include air, filtered or treated air, or other gases.
• non-permeable gas-permeable
• porous can be used to describe properties (e.g., material properties, characteristics, parameters) of one or more features of a cell culture surface.
• a non-permeable cell culture surface e.g., material of a non-permeable cell culture surface
• a non-permeable cell culture surface 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 cell culture surface under normal conditions.
• a non-permeable cell culture surface can form a portion of the wall of the vessel.
• the cell culture chamber of the vessel is considered to be sterile when a non-permeable cell culture surface forms a portion of the wall of the vessel because bacteria, for example, cannot pass through the non-permeable cell culture surface.
• gas can become trapped within the microcavity of a non-permeable cell culture surface based on surface tension of the liquid, thereby, in some embodiments, preventing material from filling the microcavities and preventing growth of a spheroid.
• a "gas-permeable" cell culture surface e.g., material of a gas-permeable cell culture surface
• a gas-permeable cell culture surface does not permit the transfer of solid and liquid in to, through, or out of, the gas-permeable cell culture surface and does permit the transfer of gas in to, through, or out of, the gas-permeable cell culture surface.
• a gas- permeable cell culture surface can form a portion of the wall of the vessel.
• the cell culture chamber of the vessel is considered to be sterile when a gas-permeable cell culture surface forms a portion of the wall of the vessel because bacteria, for example, cannot reasonably pass through the gas-permeable cell culture surface.
• gas can still become trapped in the microcavity during filling with material because gas-permeation rates through the gas-permeable cell culture surface 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 cell culture surface.
• slowly filling the microcavities allows the liquid front to enter each microcavity at an angle, thereby displacing gas as the liquid fills the microcavity.
• gas after filling the cavity with liquid, gas can permeate (slowly) through the gas-permeable cell culture surface.
• a porous cell culture surface e.g., material of a porous cell culture surface
• a porous cell culture surface does not permit the transfer of solid in to, through, or out of, the porous cell culture surface and does permit the transfer of liquid and gas in to, through, or out of, the porous cell culture surface.
• a porous cell culture surface cannot form a portion of the vessel because bacteria can pass through a porous cell culture surface, thus causing sterility issues in the cell culture chamber.
• the cell culture surface when using a porous cell culture surface, the cell culture surface 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 cell culture surface. Thus, filling of the microcavities can be performed rapidly without concern for entrapping gas in the microcavities.
• liquid can only pass through the porous cell culture surface with added pressure or physical contact and disturbance of the cell culture surface.
• material including liquid can be contained in the microcavities of the cell culture surface so long as the cell culture surface is not exposed to added pressure or physical contact and disturbance.
• the porous cell culture surface can be supported in the cell culture chamber to allow gas to pass through the cell culture surface during filling as well as during culturing and to isolate the cell culture surface from added pressure or physical contact and disturbance from external forces (e.g., outside the cell culture chamber).
• the disclosure provides a cell culture vessel comprising: a cell culture surface consisting essentially of a plurality of microcavities; a wall attached to the cell culture surface, the cell culture surface and an inner surface of the wall define a cell culture chamber of the vessel.
• each microcavity of the plurality of microcavities comprises a concave bottom and an opening.
• the disclosure provides the cell culture vessel of aspect 1 or 2, further comprising a necked opening.
• the disclosure provides the cell culture vessel of aspect 3 further comprising and dam in the necked opening.
• the disclosure provides the cell culture vessel of aspect 1 or 2 further comprising a lid.
• the disclosure provides the cell culture vessel of aspect 3 or 4 further comprising a lid.
• the disclosure provides the cell culture vessel of aspect 6 wherein the lid comprises a hinged opening.
• the disclosure provides the cell culture vessel of aspect 4 wherein the lid comprises a sliding opening.
• the disclosure provides the cell culture vessel of aspect 3, 4, or 6-8 wherein the necked opening comprises a bend.
• the disclosure provides the cell culture vessel of any one of aspects 1-9 wherein the wall comprises a recess and the cell culture surface is attached to the recess.
• the disclosure provides the cell culture vessel of any one of aspects 1-9 wherein the wall comprises a protrusion and the cell culture surface is attached to the protrusion.
• the disclosure provides a cell culture vessel comprising: a cell culture surface comprising a plurality of microcavities, the microcavities having a non- flat sinusoidal shape; a wall attached to the cell culture surface, the cell culture surface and an inner surface of the wall define a cell culture chamber of the vessel; wherein the cell culture surface is substantially free of flat areas.
• each microcavity of the plurality of microcavities comprises a concave bottom and an opening.
• the disclosure provides the cell culture vessel of aspect 12 or 13, further comprising a necked opening.
• the disclosure provides the cell culture vessel of aspect 14 further comprising and dam in the necked opening.
• the disclosure provides the cell culture vessel of aspect 12 or 13 further comprising a lid.
• the disclosure provides the cell culture vessel of aspect 14 or 15 further comprising a lid.
• the disclosure provides the cell culture vessel of aspect 17 wherein the lid comprises a hinged opening.
• the disclosure provides the cell culture vessel of aspect 17 wherein the lid comprises a sliding opening.
• the disclosure provides the cell culture vessel of any one of aspects 14, 15, or 17-19 wherein the necked opening comprises a bend.
• the disclosure provides the cell culture vessel of any one of aspects 12-17 wherein the wall comprises a recess and the cell culture surface is attached to the recess.
• the disclosure provides the cell culture vessel of any one of claims 12-17 wherein the wall comprises a protrusion and the cell culture surface is attached to the protrusion.
• Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, embodiments include from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

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• 2018
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