JP2020527345A - Cell culture vessel - Google Patents

Abstract

The cell culture vessel has a wall and a cell culture surface having a plurality of microcavities for culturing cells in a three-dimensional structure, which is called a "spheroid". The inner surface of the wall and the cell culture surface thereof define the cell culture chamber of the container. The wall is on the cell culture surface in such a way that the vessel does not provide a flat surface on or around the cell culture surface so that the vessel provides a suitable environment for the production of a three-dimensional cell culture, a homogeneous population of spheroids. It is attached.

Description

Description of related application

This application is based on its contents and is cited in its entirety here, entitled "Cell Culture Container and Methods of Culturing Cells", US Provisional Patent Application No. 62/642427 filed on March 18, 2018. US Provisional Patent Application No. 62/532681 filed on July 14, 2017, entitled "Cell Culture Container and Methods of Culturing Cells"; 7 of 2017, entitled "Cell Culture Container and Methods of Culturing Cells" US Provisional Patent Application No. 62/532639 filed on 14th March; US Provisional Patent Application No. 62/532648 filed on 14th July 2017, entitled "Cell Culture Container and Methods of Culturing Cells"; And claims the benefit of the priority of US Provisional Patent Application No. 62/532671 filed on July 14, 2017, entitled "Cell Culture Container and Methods of Culturing Cells".

The present disclosure broadly relates to a cell culture vessel and a method for culturing cells, and more particularly to a cell culture vessel for accommodating three-dimensional cells and a method for culturing three-dimensional cells in the cell culture vessel.

It is known to house three-dimensional cells in a cell culture vessel. It is also known to culture three-dimensional cells in a cell culture vessel.

The following is a brief overview of the present disclosure to provide a basic understanding of some of the exemplary embodiments described in the detailed description.

In some embodiments, the cell culture vessel has side walls and a bottom surface. In embodiments, the bottom surface is a cell culture surface with multiple microcavities. In an embodiment, the cell culture surface is a substrate attached to its side wall. In embodiments, the sidewalls are attached to the substrate so that there is no flat surface around the cell culture surface.

In embodiments, the container may comprise a top, bottom, side wall, necked opening or opening and an end wall opposite the necked opening. Alternatively, in embodiments, the cell culture vessel may have a lid. Alternatively, in embodiments, the cell culture vessel may have a lid and a necked opening or opening, which may be the top of the vessel. The inner surface of the bottom of the container is, in embodiments, the cell culture surface. The cell culture surface can span the length of the cell culture chamber. The vessel may include a neck portion of the inner surface of the wall that extends at an angle from its opening to the cell culture surface. The method of culturing cells in the cell culture vessel may include passing a liquid through the opening from outside the vessel into the cell culture chamber, thereby supplying a predetermined amount of liquid to the cell culture chamber.

In an embodiment, each microcavity of the plurality of microcavities has a concave bottom surface and an opening at the top. The liquid enters each microcavity through an opening at the top of each microcavity.

In embodiments, the container may have a necked opening or opening that can be closed with a cap. In embodiments, the top wall of the container may be a lid. In embodiments, the lid can be opened by a sliding opening, or a hinged opening, or any other known opening mechanism. In embodiments, the container does not have a necked opening and instead has a lid.

The method of culturing cells in the cell culture vessel includes a step of introducing a predetermined amount of liquid such as a liquid medium containing cells into the cell culture chamber, and the method of culturing the cells in at least one microcavity of a plurality of microcavities. It may include the step of depositing at least a portion of a fixed amount of liquid. The method may further comprise the step of depositing at least a portion of the predetermined amount of liquid in the at least one microcavity and then culturing the cells in at least one of the plurality of microcavities.

The previous embodiments are exemplary and are provided in any combination or alone with any one or more embodiments given herein, without departing from the scope of the present disclosure. There is no problem. In addition, both the general description above and the detailed description below provide embodiments of the present disclosure to understand the nature and characteristics of embodiments as described and claimed. It should be understood that there is an intention to provide an overview or gist to do so. The accompanying drawings are included to give a further understanding of the embodiments, are included in the specification and constitute a part thereof. The drawings show various embodiments of the present disclosure and serve to explain their principles and operation, along with explanations.

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

Side view of the cell culture vessel according to the embodiment of the present disclosure Top view of the cell culture vessel along line 2-2 of FIG. 1 according to the embodiment of the present disclosure. Sectional view of the cell culture vessel along line 3-3 of FIG. 2 according to the embodiment of the present disclosure. Sectional view of the cell culture vessel along line 4-4 of FIG. 1 according to the embodiment of the present disclosure. Enlarged schematic view of the cell culture vessel taken in area 5 of FIG. 4, including a cell culture surface having a plurality of microcavities, according to an embodiment of the present disclosure. 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 according to an embodiment of the present disclosure. Cross-sectional view of an alternative embodiment of a cell culture vessel comprising a cell culture surface having a plurality of microcavities in FIG. 6 according to an embodiment of the present disclosure. Partial cross-sectional view of a cell culture vessel along line 8-8 of FIG. 2 with a lid portion and a groove according to an embodiment of the present disclosure. Partial sectional view of a cell culture vessel along line 9-9 of FIG. 2 with a lid and hinges according to an embodiment of the present disclosure. Side view of three cell culture vessels stacked together according to the embodiment of the present disclosure. Sectional drawing of an alternative embodiment of the cell culture vessel of FIG. 3, comprising a method of culturing cells in a fifth exemplary cell culture vessel according to an embodiment of the present disclosure. Sectional drawing which shows the process of the method of culturing cells in the cell culture container of FIG. Enlarged schematic view of a cell culture vessel taken in area 13 of FIG. 12 including a cell culture surface having a plurality of microcavities according to an embodiment of the present disclosure. Sectional drawing which shows the process of the method of culturing cells in the cell culture container of FIG. According to an embodiment of the present disclosure, a cell culture vessel taken in area 15 of FIG. 14 having a cell culture surface having a plurality of microcavities, and cells in at least one of the plurality of microcavities. Enlarged schematic view of the method of culturing Sectional drawing which shows the process of the method of culturing cells in the cell culture container of FIG. 4 by embodiment of this disclosure Side view of the cell culture vessel according to the embodiment of the present disclosure Top view of the cell culture vessel along lines 18-18 of FIG. 17 according to the embodiment of the present disclosure. Sectional drawing of embodiment of embodiment of cell culture vessel with recess along line 19-19 of FIG. 18 according to embodiment of the present disclosure. Sectional view of the embodiment of the embodiment of the present disclosure, which is an alternative to the cell culture vessel of FIG. 19 having a recess. Enlarged schematic view of a portion of a third exemplary cell culture vessel taken in area 21 of FIG. 19 with a cell culture surface comprising a plurality of microcavities disposed within a recess according to an embodiment of the present disclosure. An enlarged schematic view of an alternative embodiment of a portion of a cell culture vessel having a cell culture surface having a plurality of microcavities arranged in a recess of FIG. 21 having a stepped portion according to an embodiment of the present disclosure. An enlarged schematic view of an alternative embodiment of a cell culture vessel having a cell culture surface including a plurality of microcavities arranged in a recess in FIG. 21 including a peripheral surface of the cell culture surface according to the embodiment of the present disclosure. Sectional view of an exemplary embodiment of a third exemplary cell culture vessel, according to an embodiment of the present disclosure, along line 24-24 of FIG. 17 having a recess. Sectional view of an exemplary cell culture vessel of FIG. 19 with protrusions according to embodiments of the present disclosure Sectional view of the cell culture vessel provided with the protrusion of FIG. 25 according to the embodiment of the present disclosure. Enlarged schematic view of a portion of the cell culture vessel taken in area 28 of FIG. 25 with a cell culture surface containing a plurality of microcavities arranged on the protrusions according to the embodiments of the present disclosure. An enlarged schematic view of a portion of a cell culture vessel comprising a cell culture surface having a plurality of microcavities arranged on a protrusion of FIG. 27 having a stepped portion according to an embodiment of the present disclosure. Cross-sectional view of the cell culture vessel of FIG. 24 provided with protrusions according to the embodiment of the present disclosure. Sectional drawing of the cell culture vessel of FIG. 19 containing a predetermined amount of liquid according to the embodiment of the present disclosure. Sectional view of the cell culture vessel containing the predetermined amount of liquid of FIG. 30 according to the embodiment of the present disclosure. An enlarged schematic view of a part of the cell culture vessel embodiment taken in area 32 of FIG. 30 including the submerged surface of the container according to the embodiments of the present disclosure. Enlarged schematic view of an alternative embodiment of a cell culture vessel comprising a submerged surface of the container of FIG. 32 having a recess according to an embodiment of the present disclosure. Enlarged schematic view of an exemplary embodiment of the embodiment of the present disclosure in place of a portion of the cell culture vessel including the submerged surface of the container of FIG. 32 with protrusions. Schematic diagram showing cells proliferating as spheroids in a microcavity and cells proliferating irregularly on a flat surface in a cell culture vessel. Schematic diagram showing cells proliferating as spheroids in a microcavity and cells proliferating irregularly on a flat surface in a cell culture vessel. Photograph of spheroids in an array of microcavities Photographs of cells proliferating in irregular cell aggregates

Here, the feature structure is more fully described below with reference to the accompanying drawings showing the exemplary embodiments of the present disclosure. Whenever possible, the same reference numbers are used throughout the drawings to refer to the same or similar parts. However, the present disclosure can be embodied in many different forms and should not be construed as limited to the embodiments described herein.

Cell culture vessels (eg, flasks) can provide a sterile cell culture chamber for culturing cells. In some embodiments, cell culture involves disease and toxicology studies, drug and therapeutic effects, tumor characteristics, organisms, genetics, and other sciences, biology, of cells and related to cells. And can provide information related to chemical principles.

In some embodiments, 3D cell cultures are more physiologically accurate than 2D cell cultures, and cells are present and grow in real-life applications compared to simulated conditions in the laboratory. It is possible to create a multicellular structure that more realistically represents the environment in which it is possible. For example, 3D cell culture has been found to provide a more rigorous realistic environment for simulating "in vivo" (ie, within living organisms in a real-life environment) cell proliferation, while 2D cell culture. Has been found to provide an environment that simulates "in vitro" (ie, intraglass) cell proliferation that does not represent the realistic environment that occurs outside the laboratory. By interacting with 3D cell cultures and observing their properties and behavior, for example, disease and toxicology studies, drug and therapeutic effects, tumors, organism characteristics, genetics, and cells and cells. An improved understanding of cells related to other scientific, biological, and chemical principles related to can be achieved. Under certain conditions, cells flock to each other to form three-dimensional "balls" called spheroids or organoids.

For these types of studies and uses, it is desirable to provide a controlled homogeneous population of spheroids. The cell culture vessel can be structured and arranged to provide a suitable environment for cells to form spheroids during culture. The cell culture vessel can include a cell culture surface containing multiple microcavities (eg, microcavities, micro-sized wells, less than milliliter-sized wells). If these microcavities are arranged in an array to provide a large number of microcavities in a single cell culture vessel, it is possible to culture a large number of spheroids, thus performing tests and experiments on a large number of cells. It will be possible to do.

However, in the presence of flat surfaces within the cell culture vessel for which spheroids are to grow, cells can accumulate on these flat surfaces to form irregular cell aggregates. These are undesirable. In embodiments, the present disclosure provides a cell culture vessel that does not have a flat surface on which cells can stack and proliferate in an irregular multicellular form. That is, the cell culture surface of the container becomes substantially from the microcavity.

In embodiments, the cell culture surface can be an insert placed in a flask, or the cell culture surface can be attached to the wall of the cell culture vessel. The cell culture surface with the array of microcavities can be bonded to the wall of the cell culture vessel by, for example, adhesion, laser welding, ultrasonic welding, or some other method. The cell culture surface can include a top side and / or a bottom side that includes a wavy (eg, sinusoidal) surface that forms multiple microcavities.

When culturing cells, the container can be filled with a material (eg, medium, solid, liquid, gas) that promotes the growth of 3D cell cultures (eg, cell aggregates, spheroids). For example, a medium containing cells suspended in a liquid can be added to the cell culture chamber of the container. Suspended cells can assemble within their multiple microcavities and form a population or mass of cells (eg, proliferate). These populations or masses are spheroids or organoids.

For example, in some embodiments, one spheroid is formed in each microcavity of multiple microcavities, at least based on gravity, and one or more cells suspended in a liquid are the liquid. It can fall through and begin to deposit within each microcavity. The shape of the microcavities (eg, the concave surface or bottom that defines the wells) and the surface coating of the microcavities that prevent cells from adhering to the surface also promote the growth of the 3D cell culture within each micro. be able to. That is, the cells form spheroids and are constrained to grow to a particular size by the size of the microcavities. During culturing, spheroids can consume medium (eg, food, nutrients) and produce metabolites (eg, waste products) as by-products. Therefore, in some embodiments, the food medium can be added to the cell culture chamber during the culture and the waste medium can be removed from the cell culture chamber during the culture. Attempts can be made to avoid moving spheroids out of the microcavities and promote the desired cell culture of spheroids when adding and removing medium.

Here, with reference to FIGS. 1 to 37, embodiments of the cell culture vessel 100 and the method of culturing cells in the cell culture vessel 100 will be described. FIG. 1 shows a side view of an embodiment of the cell culture vessel 100, and FIG. 2 shows a plan view of the vessel 100 along line 2-2 of FIG. In some embodiments, the cell culture vessel 100 can include a port shown in FIG. 1 covered with a top 101, a bottom 108, a necked opening 112 and a cap 104. FIG. 2 in plan view shows a wall 107 surrounding the cell culture surface 115. Each of these characteristic structures of the top 101, bottom 108, and wall 107 (shown in FIG. 2) and necked opening 112 has an internal surface. That is, the top 101 has an interior surface 201, the wall 107 has an interior surface 207, the bottom 108 has an interior surface 208, and the necked opening 112 has an interior surface 212. These internal surfaces define the cell culture chamber 103. The inner surface 208 of the bottom 108 of the container 100 is the cell culture surface 115. The cell culture surface 115 has an array of microcavities (see FIGS. 5-7) for containing and culturing spheroids. As shown in FIG. 2, the inner surface of the wall is in contact with the cell culture surface 115. In embodiments, there is no flat surface between the cell culture surface 115 and the inner surface 207 of the wall 107. That is, the cell culture surface 115 is substantially free of flat surfaces. In other words, the cell culture surface 115 is made entirely of microcavities. The cell culture surface becomes substantially from the microcavity.

The container can be made from materials including, but not limited to, polymers, polycarbonate, glass, and plastics. In the drawings, the container 100 is shown to be made from a clear (eg, transparent) material; however, in some embodiments, the container 100 is, as another method, the scope of the present disclosure. May be manufactured from translucent, almost opaque, or opaque materials without departing from. FIG. 3 shows a cross-sectional view of the container 100 along line 3-3 of FIG. In some embodiments, the cell culture surface 115 and the inner surface 207 of the wall 107 define the cell culture chamber 103 of the container 100, and the opening 105 passes through the wall 107 to the cell culture chamber 103 and the fluid. Communicating. For example, in some embodiments, the cell culture chamber 103 may include the interior space volume of the container 100.

Returning to FIGS. 1 and 2, in some embodiments, the container 100 covers the opening 105 and either seals or closes the opening 105, thereby passing through the opening 105. A cap 104 properly placed so as to block the passage from the outside of the container 100 to the cell culture chamber 103 can be provided. For clarity, the cap 104 has been removed and therefore, although not shown in other drawings, in some embodiments, the cap 104 is provided and the container 100 is provided without departing from the scope of the present disclosure. It should be understood that it can be selectively added to or removed from the opening 105 of the. In some embodiments, the cap 104 can include a filter that allows the transfer of gas into and / or from the cell culture chamber 103 of the container 100. For example, in some embodiments, the cap 104 regulates the pressure of the gas in the cell culture chamber 103, thereby adjusting the cell culture chamber to the pressure of the environment outside the container 100 (eg, atmospheric pressure). A gas permeable filter properly placed to prevent pressurization (eg, overpressure) of 103 can be provided.

In some embodiments, cell cultures are shown in FIG. 3, which shows a cross-sectional view along line 3-3 of FIG. 2, and FIG. 4, which shows a cross-sectional view of FIG. 1 along line 4-4. The surface 115 can span the length "L1" of the cell culture chamber extending along the axis 510. FIG. 5 shows an enlarged schematic view of a portion of the cell culture surface 115 taken in area 5 of FIG. Further, FIG. 6 shows a partial cross-sectional view 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. .. As shown in FIG. 5, in some embodiments, the microcavities 120 can be arranged diagonally, but in other embodiments, other arrangements may be provided. Further, in some embodiments, each microcavity 120a, 120b, 120c may include concave bottoms 121a, 121b, 121c (see FIGS. 6 and 7) defining wells 122a, 122b, 122c. .. Further, each of the microcavities 120a, 120b, 120c can be provided with openings 123a, 123b, 123c at the top of each microcavity 120. As shown in FIG. 6, in some embodiments, the first surface 125 of the cell culture surface 115 can comprise a non-linear (eg, wavy, sinusoidal) profile and is the second of the cell culture surface 115. The surface 126 can be flat. Similarly, as shown in FIG. 7, in some embodiments, both the first side 125 and the second side 126 of the cell culture surface 115 are provided with a non-linear (eg, wavy, sinusoidal) profile. Can be done.

In some embodiments, the cell culture surface 115, as well as the containers 100 (as described in FIGS. 1-16) and 300 (as described in FIGS. 17-34), are polystyrene, but not limited to: , Polymethylmethacrylate, Polyvinyl Chloride, Polypropylene, Polysulfone, Polystyrene Polymeric, Fluoropolymer, Polyester, Polypolymer, Polystyrene-butadiene Copolymer, Fully Hydrogenated Styrene Polymer, Polycarbonate PDMS Copolymer, and Polyethylene, It may include polymeric materials including polyolefins such as polypropylene, polymethylpentene, polypropylene copolymers and cyclic olefin polymers. In addition, in some embodiments, at least a portion of the wells 122a, 122b, 122c defined by the concave bottoms 121a, 121b, 121c is coated with an ultra-low bond coating, whereby the wells 122a, At least a portion of 122b, 122c can be non-adhesive to cells. For example, in some embodiments, one or more of a perfluoropolymer, a nonionic hydrogel such as olefin, agarose, polyacrylamide, a polyether such as polyethylene oxide, a polyol such as polyvinyl alcohol, or a mixture thereof is concave. It can be applied to at least a part of the wells 122a, 122b, 122c defined by the bottoms 121a, 121b, 121c.

Further, in some embodiments, the microcavities 120a, 120b, 120c of the plurality of microcavities 120 (as described in FIGS. 1-16) and 320 (as described in FIGS. 17-34) are Various feature structures and variants of those feature structures can be included without departing from the scope of the present disclosure. For example, in some embodiments, the plurality of microcavities 120 may be arranged in an array that includes a linear arrangement (shown), a diagonal arrangement, a rectangular arrangement, a circular arrangement, a radial arrangement, a hexagonal close-packed arrangement, and the like. it can. In addition, in some embodiments, the openings 123a, 123b, 123c can include various shapes. In some embodiments, the openings 123a, 123b, 123c can include one or more of circular, oval, rectangular, quadrilateral, hexagon, and other polygons. In addition, in some embodiments, the openings 123a, 123b, 123c can include dimensions from about 100 micrometers (μm) to about 5000 μm (eg, diameter, width, square or rectangular diagonal, etc.). .. For example, in some embodiments, the openings 123a, 123b, 123c are 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, It can include dimensions of 900 μm, 950 μm, 1000 μm, 1500 μm, 2000 μm, 2500 μm, 3000 μm, 3500 μm, 4000 μm, 4500 μm, 5000 μm, and any size or range of dimensions within the range of about 100 μm to about 5000 μm.

In some embodiments, wells 122a, 122b, 122c (as described in FIGS. 1-16) and 322 (as described in FIGS. 17-34) defined by concave bottoms 121a, 121b, 121c. ) Can include various shapes. In some embodiments, the wells 122a, 122b, 122c defined by the concave bottoms 121a, 121b, 121c are circular, oval, parabolic, hyperbolic, chevron, inclined, or other cross-sections. It can include one or more profile shapes. In addition, in some embodiments, the depths of the wells 122a, 122b, 122c (eg, the depth from the plane defined by the openings 123a, 123b, 123c to the concave bottoms 121a, 121b, 121c). Can include dimensions from about 100 micrometers (μm) to about 5000 μm. For example, in some embodiments, the depths of wells 122a, 122b, 122c are 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm. , 850 μm, 900 μm, 950 μm, 1000 μm, 1500 μm, 2000 μm, 2500 μm, 3000 μm, 3500 μm, 4000 μm, 4500 μm, 5000 μm, and any size or range of dimensions contained within the range of about 100 μm to about 5000 μm. it can.

In some embodiments, three-dimensional cells 150 (eg, spheroids, organoids 150a, 150b, 150c) that can be cultured in at least one microcavity 120a, 120b, 120c of a plurality of microcavities 120 (see FIG. 16) (see FIG. (As described in FIGS. 1-16) and 350 (as described in FIGS. 17-34) are included in dimensions (eg, diameter) of about 50 μm to about 5000 μm, and within the range of about 50 μm to about 5000 μm. It can include any dimension or range of dimensions. In some embodiments, dimensions larger or smaller than the disclosed explicit dimensions can be provided, and therefore, unless otherwise stated, dimensions larger or smaller than the disclosed explicit dimensions are those of the present disclosure. It is considered to be within the range. For example, in some embodiments, one or more dimensions of openings 123a, 123b, 123c, depths of wells 122a, 122b, 122c, and dimensions of three-dimensional cells 150 (eg, spheroids 150a, 150b, 150c). May be larger or smaller than the stated explicit dimensions disclosed, without departing from the scope of the present disclosure.

Returning to FIGS. 1-4, in some embodiments, the container 100 may comprise a neck 555 extending from the opening 105 to the cell culture surface 115. In some embodiments, the neck 555 has an inclined (eg, oblique) profile, a profile that narrows towards and / or away from the opening 105, and towards and / or away from the cell culture surface 115. It can include one or more profiles that spread in the direction. As shown in FIG. 3, in some embodiments, the neck 555 is angled with respect to the cell culture chamber 103. Further, in some embodiments, the neck 555 of the container may have a bend 158. In some embodiments, the bend 158 is higher than the cell culture surface 115. However, it should be understood that in some embodiments, the bend can be of any shape. For example, the bend 158 can be curved, inclined, or angled, or stepped.

In addition to, or instead, as shown in FIGS. 3 and 4, in some embodiments, the cell culture vessel 100 can comprise a dam 130 extending from the internal surface 212 of the neck 112. .. In some embodiments, the dam 130 may include a surface 131 facing a port that blocks a passage defined between the opening 105 and the cell culture surface 115. In some embodiments, the surface 131 facing the port of the dam 130 may be substantially perpendicular to the axis 510 of the vessel 100. Further, as shown in FIG. 3, in some embodiments, at least a portion of the free end 135 of the dam 130 can be located at a distance "d10" from the inner surface 201 of the top 101. .. In some embodiments, by disposing at least a portion of the free end 135 of the dam 130 at a distance from the inner surface 201, in some embodiments, the rear portion of the container 100 (eg, the opening 105). Access to the opposite side) can be provided. For example, in some embodiments, one or more instruments (not shown) are inserted across the dam 130 (eg, through the distance "d10") into the opening 105 of the container 100. An area of cell culture chamber 103 located behind the dam 130 can be reached. Thus, for example, as disclosed with respect to the dam 130 of the cell culture vessel 100 of the first example, in some embodiments, the dam 130 of the cell culture vessel 100 is similarly a material that flows into the vessel 100 ( For example, food, nutrients) and / or material (eg, waste products) spilling out of container 100 into the cell culture chamber 103 of container 100 while at least one of slowing down and blocking it. Bulk access can also be enabled.

As shown in FIG. 1, in some embodiments, the container 100 can include a lid 137. In some embodiments, the lid 137 can be the top 101 of the container 100 when closed (see also FIG. 3). In some embodiments, the first main surface 538 can define at least a portion of the cell culture chamber 103. In addition, in some embodiments, the lid 137 can be opened and closed, or opened to some extent (or closed to some extent). The lid 137 can be slidably attached to the container 100, or in some embodiments, the lid 137 can be hinged to the container 100. In some embodiments, the opening 536 can fluidly communicate with the cell culture chamber 103.

Further, as shown in FIG. 3, in some embodiments, the first distance "d11" from the cell culture surface 115 to the top 101 or lid 137 is the opening 507 from the cell culture surface 115 to the opening 105. It can be less than the second distance "d12" to. In some embodiments, the second distance "d12" from the cell culture surface 115 to the opening 507 of the opening 105 may be defined at any position in the opening 507. However, in some embodiments, the second distance "d12" from the cell culture surface 115 to the opening 507 of the opening 105 is defined as the closest position of the opening 105 opening 507 to the cell culture surface 115. be able to. For example, in some embodiments, when the container 100 is oriented so that the axis 510 extends in a direction perpendicular to the direction of gravity "g" (see FIG. 16), from the cell culture surface 115. The second distance "d12" of the opening 105 to the opening 507 can be defined as the lowest position of the opening 507 of the opening 105 with respect to the direction of gravity "g".

Thus, in some embodiments, for example, with a comparative vessel in which the distance extending from the cell culture surface 115 to the portion of the lid 137 is greater than the second distance from the cell culture surface 115 to the opening 507 of the opening 105. In comparison, one or more feature structures of the container 100 can provide a cell culture chamber 103 containing a larger volume that can accommodate the material, alone or in combination. That is, when the container 100 is oriented so that the axis 510 extends in a direction perpendicular to the direction of gravity "g", the second distance "from the cell culture surface 115 to the opening 507 of the opening 105" "d12" (defined as the lowest position of opening 507 of opening 105 relative to the direction of gravity "g") defines the maximum filling line for the volume of material that can be accommodated in the cell culture chamber 103 of container 100. be able to. For example, if the second distance "d12" is smaller than the first distance "d11" and the container 100 is oriented so that the axis 510 extends in a direction perpendicular to the direction of gravity "g". The maximum filling line for the volume of material contained within the cell culture chamber 103 will correspond to the distance from the lid 137. This is because any additional material added to the cell culture chamber 103 will flow out of the opening of the opening 105 without being housed in the cell culture chamber 103. Therefore, when the second distance "d12" is smaller than the first distance "d11" and the container 100 is oriented so that the axis 510 extends in a direction perpendicular to the direction of gravity "g". , Container 100 may include a volume containing a portion of the cell culture chamber 103 that is not utilized for the material it contains. Thus, in some embodiments, the entire cell culture chamber 103 is provided by providing a container 100 according to an embodiment of the present disclosure, comprising a second distance "d12" greater than a first distance "d11". The product can be used to contain material and a larger volume of material can be contained within the cell culture chamber 103 to achieve efficient distribution of material and total utilization of space in container 100. be able to. Similarly, in some embodiments, the second distance "d12" may be equal to the first distance "d11" without departing from the scope of the present disclosure.

Further, in some embodiments, with respect to the unit area of the cell culture surface 115 (eg, the unit area providing each surface capable of culturing one or more types of cells), the three-dimensional cell culture is, for example, comparatively two. It consumes more medium (eg, food, nutrients) than dimensional cell culture and can produce more medium (eg, waste products) as a by-product. Therefore, in some embodiments, as compared to, for example, comparative two-dimensional cell cultures, three-dimensional cell cultures according to the embodiments of the present disclosure have more frequent medium exchanges (eg, food) over an equivalent period. , Addition of nutrients, and / or removal of waste products). In addition to or instead, in some embodiments, for example, compared to comparative two-dimensional cell cultures, three-dimensional cell cultures according to the embodiments of the present disclosure have a larger medium volume over an equivalent period. (For example, it consumes more food, nutrients, and / or produces more waste products). Therefore, in some embodiments, the one or more characteristic structures of the cell culture vessel 100, and the method of culturing the cells 150 in the cell culture vessel 100, are the frequency of medium exchange and the cell culture chamber 103 of the cell culture vessel 100. It provides an advantage in terms of medium volume, which can be one or more that can be contained within, added to the cell culture chamber 103, and removed from the cell culture chamber 103, thereby culturing three-dimensional cells. It can provide a desirable and effective environment.

As shown in FIG. 8, which shows a partial cross-sectional view of the container 100 along line 8-8 of FIG. 2, in some embodiments, the top wall 101 can include a groove 180 and the lid 137. For example, it may be slidable within the groove 180 (as shown by arrow 181 in FIG. 2) to selectively provide access to the cell culture chamber 103 through the opening of the opening 136. In addition to or instead, in some embodiments, the container 100 has a lid 137 walled, as shown in FIG. 9, which shows a partial cross-sectional view of the container 100 along line 9-9 of FIG. A hinge 182 connected to 101 can be provided. In some embodiments, the lid 137 may be rotatable around the hinge 182 to selectively provide access to the cell culture chamber 103, eg, through the opening of the opening 136 (arrow 183). As shown by). Further, in some embodiments, the lid 137 is a reusable adhesive and a non-reusable adhesive to selectively provide access to the cell culture chamber 103, for example through the opening of the opening 136. It may be connected to the wall 101 with an adhesive containing an agent (not shown) and / or one or more feature structures (not shown).

In addition, in some embodiments, the length "L2" of the container 100, measured along the axis 510 of the container from the port 105 to the end wall 107, is the length "L1" of the cell culture surface 115. It can be more than that. Thus, in some embodiments, the plurality of containers 100 can be stacked (eg, gravity) in order to reduce, for example, the surface area occupied by the plurality of containers 100 (eg, laboratory surface area, table surface area). (Vertical to the direction of). For example, FIG. 10 schematically shows a side view of a plurality of containers 100, 100a, 100b stacked with each other according to the embodiment of the present disclosure.

As outlined in FIG. 10, in some embodiments, a container 100a with a wall 101a, a cap 104a, a neck 555a, a lid 137a, and a bottom 108a is stacked on top of another. be able to. For example, in some embodiments, the bottom 108b of the container 100b can be placed on a horizontal plane (not shown) that defines a main plane perpendicular to the direction of gravity with respect to the walls 101, 101a, 101b. .. In some embodiments, the bottom 108 of the container 100 can be placed on (eg, facing) the lid 137b of the container 100b. Similarly, in some embodiments, the bottom 108a of the container 100a can be placed on (eg, facing) the lid 137 of the container 100. By arranging (eg, stacking) the plurality of containers 100, 100a, 100b according to the embodiments of the present disclosure, the plurality of containers 100, 100a, 100b can be stored, washed, for example, with respect to the containers 100, 100a, 100b. And during one or more of culturing, the space (eg, area, volume) in which the containers 100, 100a, 100b are provided can be efficiently utilized. Although the three stacked containers 100, 100a, 100b are shown as plural, two containers or four or more containers are stacked according to the embodiments of the present disclosure without departing from the scope of the present disclosure. It should be understood that Moreover, in some embodiments, the plurality of containers 100, 100a, 100b are separately, in various arrangements, including arrangements not expressly disclosed in the present disclosure, without departing from the scope of the present disclosure. And / or can be placed together (eg, stacked).

In embodiments, the containers are stacked. Further, access to ports 105, 105a, 105b is a plurality of containers when stacked according to embodiments of the present disclosure, at least in part based on one or more feature structures of the bend 158 at the neck 112. While the 100, 100a, 100b are stacked (eg, fixed), eg, add material (eg, food, nutrients) to, for example, the cell culture chambers 103, 103a, 103b, and / or from there. It can be maintained to remove material (eg, waste products). For example, in some embodiments, stacking containers that do not have one or more of the feature structures of the present disclosure results in one or more limiting, blocking, and blocking access to the opening of the opening. obtain. In some embodiments, stacked containers containing an opening opening in which one or more of the access is restricted, blocked, and hindered provide access to the opening opening, for example. In order to do so, they may be moved relative to each other during the culture process (eg, at least one of translation and rotation). However, in some embodiments, moving the vessels relative to each other during the culture process results in one or more of the cells being cultured in the vessel being pushed away and disturbed, thereby causing. , Will adversely affect the cell culture process. Thus, ports when stacked according to embodiments of the present disclosure, based at least to some extent on one or more characteristic structures of the necks 555, 555a, 555b of the containers 100, 100a, 100b and the bends 158, 158a, 158b. Access to the openings can be achieved and benefits can be gained with respect to the cell culture process.

Here, a method of culturing cells in the cell culture vessel 100 will be described with reference to FIGS. 11 to 16. As shown in FIG. 11, in some embodiments, the method of culturing cells 150 (see FIG. 15) in the cell culture vessel 100 is through the opening 105 into the cell culture chamber 103 from outside the vessel 100. It may include the step of passing the liquid through (eg, indicated by the arrow 106), thereby providing a predetermined amount of the liquid 140 into the cell culture chamber 103. In embodiments, the method can be performed in a container with or without the optional dam 130.

Also shown is a bent portion 158 of the neck 112 of the container 100. In some embodiments, the method may include tilting the container 100 such that the bend 158 forms the lowest point at the neck 112. The liquid 140 introduced into the container can accumulate in the neck 112 at the bend 158. That is, the container can contain a predetermined amount of the liquid 140 in the bent portion 158 of the neck 112 without the liquid 140 coming into contact with the array 115 of the microcavities. As described in more detail below, by preventing the liquid 140 from contacting one or more microcavities 120 of the cell culture surface 115 containing the array of microcavities 120, eg, cells, at this stage of the method. It can provide some advantages to promote improved culture of 150 (see FIG. 15). The axis 510 is then perpendicular to gravity "g" so that the liquid 140 slowly flows across the cell culture surface 115 with the array of microcavities 120 so that the liquid slowly fills the microcavities 120. In addition, the container can be tilted slowly. This process is outlined in FIG.

For example, FIG. 13 shows an enlarged schematic view of the cell culture vessel 100 taken in area 13 of FIG. 12, where at least a portion of the liquid 140 extends from the neck 112 to the length "L1" of the cell culture chamber 103. It is shown to flow along the cell culture surface 115 and enter the microcavities 120a, 120b, 120c of the array of microcavities 120. In some embodiments, the operation of the container 100 flowing the liquid can be controlled and can be slow (eg, done over a period of approximately minutes). For example, it has been observed that when the microcavities 120a, 120b, 120c of a plurality of microcavities 120 are directly filled with a liquid (for example, not based on the method of the present disclosure), bubbles can be formed in the microcavities. This slow introduction of liquid into the microcavity 120 reduces the formation of bubbles and allows the liquid to flow into the microcavity. Bubbles interfere with cell growth. The liquid medium can have high surface tension (the liquid medium can be highly viscous) and the microcavities 120 are very small. Although not intended to be bound by theory, if the microcavities 120a, 120b, 120c are attempted to be filled directly and rapidly with liquid (eg, for a period of approximately several seconds), due to surface tension, air bubbles will form in the microcavities. It is thought that it will be captured within. However, by utilizing one or more feature structures of the methods of the present disclosure, for example, a liquid is introduced into the bent portion 158 of the neck 112, and then the container 100 is placed in its cell culture position (the cell culture surface is gravity "g". It is observed that the formation of air bubbles can be reduced by slowly tilting (perpendicular to).

In addition, for long-term cell cultures, the medium must be changed to ensure that the cells maintain a fresh supply of nutrients. This requires removing and replenishing the medium while the spheroids are in place within each microcavity 120. It is important not to push the spheroids out of the microcavity 120 during the medium change. If the spheroid 150 "jumps out" of its microcavity 120, it can sink into another already occupied microcavity. When the spheroids touch each other, they form an irregular cell assembly 801 (see FIGS. 35 and 36), resulting in heterogeneous cell culture.

In some embodiments, the bend 158 of the neck 112 may abut on the cell culture surface 115, allowing the liquid 140 to flow from the bend 158 of the neck 112 in a controlled flow (eg, reduced liquid droplets). With or without turbulence (with or without reduced turbulence), deposits in the microcavities 120a, 120b, 120c, thereby moving the gas from the wells 122a, 122b, 122c while moving the microcavities 120a, 120b, A steady flow of liquid deposited in the wells 122a, 122b, 122c of the microcavities 120a, 120b, 120c can be provided through a portion of the respective openings 123a, 123b, 123c of 120c. In embodiments, the cell culture surface 115 extends from wall 107 to wall 107. In embodiments, the cell culture surface 115 does not have any flat area. That is, the cell culture surface is an array of wall-to-wall microcavities with no edges or flat areas between the cell culture surface and the wall 107. In embodiments, the cell culture surface is substantially composed of multiple microcavities. In embodiments, there is no flat area in the cell culture chamber where the cells settle. This is important to ensure that the cells do not colonize the cell culture chamber outside the microwell. When cells settle in a flat area outside the cell culture surface outside the microwell, the cells can proliferate as irregular cell aggregates and form heterogeneous populations of multicellular 3D structures within their vessels. In embodiments, the cell culture surface is substantially composed of multiple microcavities. When cells colonize a flat area outside the cell culture surface outside the microwell, the cells proliferate as irregular cell aggregates 801 (see Figures 35A and 35B, 36A and 36B) and are multicellular in their vessels. It can form heterogeneous populations of 3D structures. In embodiments, the cell culture surface is substantially composed of multiple microcavities.

In embodiments, the cell culture surface 115 extends from wall 107 to wall 107. In embodiments, the cell culture surface 115 does not have any flat area. That is, the cell culture surface is an array of microcavities 120 extending wall-to-wall with no edges or flat areas between the cell culture surface and the wall 107. In embodiments, the cell culture surface is substantially composed of multiple microcavities. In embodiments, there is no flat area in the cell culture chamber where the cells settle. This is important to ensure that the cells do not colonize the cell culture chamber outside the microwell. When cells colonize a flat area outside the cell culture surface outside the microwell, the cells proliferate as irregular cell aggregates 801 (see Figures 35A and 35B, 36A and 36B) and are multicellular in their vessels. It can form heterogeneous populations of 3D structures. In embodiments, the cell culture surface is substantially composed of multiple microcavities.

As shown in FIG. 14, in some embodiments, the liquid 140 can be flushed from the neck 112 over the entire surface of the cell culture surface 115, at least based on the movement of the container 100. In addition, FIG. 15 shows an enlarged schematic view of the cell culture vessel 100 taken in area 15 of FIG. 14, including a method of culturing the cells 150 in the cell culture vessel 100. For example, in some embodiments, the method deposits at least a portion of a predetermined amount of liquid 140 in at least one microcavity 120a, 120b, 120c and then at least one of the plurality of microcavities 115. A step of culturing cells 150 (eg, spheroids 150a, spheroids 150b, spheroids 150c) in the microcavities 120a, 120b, 120c can be included. As shown in FIGS. 14 and 15, in some embodiments, the axis 510 of the container 100 is substantially relative to the direction of gravity "g" while culturing the cells 150 in the microcavity 120. Can be vertical.

As outlined in FIG. 16, in some embodiments, the method can further include adding additional liquid medium 140 to the cell culture chamber 103 (as indicated by arrow 508). For example, in some embodiments, while culturing cells 150 (see FIG. 15) in cell culture vessel 100, liquid medium 140 (liquid containing cell food, nutrients) is placed in cell culture chamber 103. Can be added to. Since the port 150 is raised and points upwards, the liquid 140 can be added to the container up to a distance "d13" from the lowest point of the opening 507 of the port 150. That is, in the embodiment, the container can be filled up to the lid 137 or the upper part 101 of the container 100. In this way, the cell culture chamber 103 can accommodate a larger volume of liquid 140 that would be accommodated if the port were located below the top 101 of the container 100.

Here, with reference to FIGS. 17 to 34, additional embodiments of the cell culture vessel 300 and the method of culturing cells in the cell culture vessel 300 are described. The embodiment shown in FIGS. 17-34 shows an embodiment without a necked opening or a port, whereas the embodiment shown in FIGS. 17-34 is a container with a port. It will be appreciated that it can be incorporated into the embodiments shown in FIGS. 1-16. For example, FIG. 17 schematically shows a side view of the cell culture vessel 300, and FIG. 18 schematically shows a plan view of the vessel 300 along the line 18-18 of FIG. In some embodiments, the cell culture vessel 300 can include a wall 301 and a lid 304. In the drawings, the container 300 is shown to be made from a clear (eg, transparent) material; however, in some embodiments, the container 300 is, as another method, the scope of the present disclosure. It can be manufactured from translucent, almost opaque, or opaque materials without departing from. In some embodiments, the lid 304 covers the opening of the container 300 and either seals or closes the opening, thereby from the outside of the container 300 through the opening to the cell culture chamber 303. Can be placed correctly so as to block the passage. For clarity, the lid 304 has been removed and therefore, although not shown in other drawings, in some embodiments, the lid 304 is provided and the container 300 is provided without departing from the scope of the present disclosure. It should be understood that it can be selectively added to or removed from the opening of the. In some embodiments, the lid 304 can include a filter (see FIG. 19) that allows the transfer of gas into and / or from the cell culture chamber 303 of the container 300. For example, in some embodiments, the lid 304 regulates the pressure of the gas in the cell culture chamber 303, thereby adjusting the cell culture chamber to the pressure of the environment outside the container 300 (eg, atmospheric pressure). A gas permeable filter properly placed to prevent pressurization (eg, overpressure) of 303 can be provided.

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 embodiment of the cell culture vessel 300 of FIG. The cross-sectional view of the form is shown. In addition, FIG. 25 shows a cross-sectional view of an alternative exemplary embodiment of the cell culture vessel 300 of FIG. 18, and FIG. 26 shows an alternative exemplary embodiment of the cell culture vessel 300 of FIG. A cross-sectional view is shown. In some embodiments, the wall 301 can comprise an inner surface 302 and the container 300 can comprise a cell culture surface 315 containing a plurality of microcavities 320. As shown in FIGS. 19 and 25, in some embodiments, the cell culture surface 315 and the inner surface 302 of the wall 301 define the cell culture chamber 303 of the container 300. Alternatively, as shown in FIGS. 20 and 26, in some embodiments, the cell culture chamber 303 of the container 300 in which the inner surface 302 of the wall 301 comprises, for example, a first region 303a and a second region 303b. The cell culture surface 315 can be positioned between the first region 303a and the second region 303b in the cell culture chamber 303.

As shown in FIGS. 19 and 20, in some embodiments, the outer circumference 330 of the cell culture surface 315 can surround a plurality of microcavities 320, and at least a portion 331 of the outer circumference 330 is a container. It can be positioned in the recess 335 of the inner surface 302 of the wall 301 of the 300. For example, FIG. 24 shows an exemplary cross-sectional view of the container 300 taken along line 24-24 of FIG. 17, where the entire perimeter 330 surrounds the plurality of microcavities 320 along the sides. The entire outer circumference 330 is positioned in the recess 335. Alternatively, as shown in FIGS. 25 and 26, in some embodiments, the outer circumference 330 of the cell culture surface 315 can surround a plurality of microcavities 320, with at least a portion 331 of the outer circumference 330 being It can be positioned on the protrusion 337 of the inner surface 302 of the wall 301 of the container 300. For example, FIG. 29 shows a cross-sectional view of an exemplary embodiment in place of the third exemplary cell culture vessel 300 of FIG. 24, where the total perimeter 330 has a plurality of microcavities 320 along the sides. The entire outer circumference 330 thereof is positioned on the protrusion 337. Throughout the present disclosure, "surrounding" means, for example, the outer circumference defined by the first feature structure in a top view or bottom view in a direction perpendicular to the main feature structure of the cell culture surface 315. , Means to surround the outer circumference defined by the second feature structure. Therefore, for example, as shown in FIGS. 24 and 29, the outer circumference of the cell culture surface 315 (defined by the outer circumference 330) is defined by the outer circumference of the cell culture surface 315 (defined by the plurality of microcavities 320). Surrounding).

In an embodiment, the cell culture surface 315 extends from wall 301 to wall 301. In embodiments, the cell culture surface 315 does not have any flat area. That is, the cell culture surface is an array of microcavities 320 extending from wall to wall with no edges or flat areas between the cell culture surface and the wall 301. In embodiments, the cell culture surface is substantially composed of multiple microcavities. In embodiments, there is no flat area in the cell culture chamber where the cells settle. This is important to ensure that the cells do not colonize the cell culture chamber outside the microwell. When cells colonize a flat area outside the cell culture surface outside the microwell, the cells proliferate as irregular cell aggregates 801 (see Figures 35A and 35B, 36A and 36B) and are multicellular in their vessels. It can form heterogeneous populations of 3D structures. In embodiments, the cell culture surface is substantially composed of multiple microcavities.

21-23 is an extension of some exemplary embodiment of the container 300 taken in area 21 of FIG. 19, including at least a portion 331 of the outer circumference 330 of the cell culture surface 315 located within the recess 335. The figure is shown. Similarly, FIGS. 27 and 28 are extensions of some exemplary embodiments of the container 300 taken in area 28 of FIG. 25, including at least a portion 331 of the outer circumference 330 located on the protrusion 337. The figure is shown. In some embodiments, each microcavity 320a, 320b, 320c of the plurality of microcavities 320 comprises concave surfaces 321a, 321b, 321c and openings 323a, 323b, 323c defining wells 322a, 322b, 322c. Can be done. Liquids enter and exit their microcavities through openings 323a, 323b, and 323c. In some embodiments, the cell culture surface 315 can be attached to the wall 301 of the container 300. For example, in some embodiments, the cell culture surface 315 is bonded (not shown), solvent (not shown), fasteners (not shown), or welded (laser welding or ultrasonic welding). It can be attached to the wall 301 of the container 300, or the wall and the cell culture surface 315 can be molded together. In addition to or instead, in some embodiments, the cell culture surface 315 is at least in part based on the operation of, for example, a plastic welding process, a laser welding process, an ultrasonic welding process, the container 300. It can be attached to the inner surface 302 of the wall 301. In some embodiments, at least one of the wall 301 and the outer circumference 330 is to promote the binding of the cell culture surface 315 to the wall 301 of the container 300, at least in part, based on the manipulation of the plastic welding process. It may include an energy director (not shown).

In addition, as shown in FIG. 22, in some embodiments, the container 300 can include a stepped portion 306 that extends outward from the cell culture surface 315 to form a recess 335. In some embodiments, the stepped portion 306 increases at least one of the volume of the cell culture chamber 303 and the quantity of the microcavities 320a, 320b, 320c of the plurality of microcavities 320 within the cell culture chamber 303. Can be done. In addition to or instead, the stepped portion 306 is compared, for example, with respect to the thickness of the corresponding recess 335 and wall 301 shown in FIG. 21 without increasing the thickness of the wall 301 of the container 300. A large recess 335 can be provided. In some embodiments, the recess 335 formed by the stepped portion 306 can be directed to accommodate at least a portion 331 of the outer circumference 330 of the cell culture surface 315 located within the recess 335. Similarly, as shown in FIG. 28, in some embodiments, the vessel 300 may comprise a stepped portion 308 extending inward towards the cell culture surface 315 to form a protrusion 337. it can. In some embodiments, the stepped portion 308 does not increase the thickness of the wall 301 of the container 300 as compared to, for example, the thickness of the corresponding protrusions 337 and the wall 301 shown in FIG. 27. A relatively large protrusion 337 can be provided. In some embodiments, the protrusion 337 formed by the stepped portion 308 can be directed to accommodate at least a portion 331 of the outer circumference 330 of the cell culture surface 315 located on the protrusion 337. ..

In an embodiment, the cell culture surface 315 extends from wall 301 to wall 301. In embodiments, the cell culture surface 315 does not have any flat area. That is, the cell culture surface is an array of microcavities 320 extending from wall to wall with no edges or flat areas between the cell culture surface and the wall 301. In embodiments, the cell culture surface is substantially composed of multiple microcavities. In embodiments, there is no flat area in the cell culture chamber where the cells settle. This is important to ensure that the cells do not colonize the cell culture chamber outside the microwell. When cells colonize a flat area outside the cell culture surface outside the microwell, the cells proliferate as irregular cell aggregates 801 (see Figures 35A and 35B, 36A and 36B) and are multicellular in their vessels. It can form heterogeneous populations of 3D structures. In embodiments, the cell culture surface is substantially composed of multiple microcavities.

In addition, as shown in FIGS. 23, 25, and 28, in some embodiments, at least a portion 331 of the outer circumference 330 of the cell culture surface 315 is each microcavity 320a of the plurality of microcavities 320. , 320b, 320c can be arranged in a direction away from the concave surfaces 321a, 321b, 321c, at intervals from the portion of the cell culture surface 315 including the openings 323a, 323b, 323c of the microcavities 320a, 320b, 320c. For example, in some embodiments, the cell culture surface 315 may comprise an outer peripheral surface 332 extending from at least a portion 331 of the outer circumference 330 to a portion of the cell culture surface 315 including openings 323a, 323b, 323c. it can. In some embodiments, the outer surface 332 can have a vertical orientation (eg, extending in the direction of gravity); however, in some embodiments, the outer surface 332 is of gravity. The cells can be oriented with respect to the direction, for example, at openings 323a, 323b, 323c of the microcavities 320a, 320b, 320c.

Further, by locating at least a portion 331 of the outer circumference 330 of the cell culture surface 315 within the recess 335, in some embodiments, for example, the opening 323a of the microcavity 320a is located on the inner surface of the wall 301 at the location of the recess 335. It can be positioned to abut the 302. For example, in some embodiments, for example, in some embodiments, the opening 323a of the microcavity 320a allows cells suspended in a liquid to adhere to or without colonization on the surface of the container 300. Instead, it may fall into wells 322a of the microcavity 320a (eg, at least based on gravity) and / or be coplanar with the inner surface 302 of the wall 301 so that it is directed by the inner surface 302. Similarly, by positioning at least a portion 331 of the outer circumference 330 of the cell culture surface 315 on the protrusion 337, in some embodiments, for example, the protrusion 337 supports the outer circumference 330 to support the microcavity 320a. The opening 323a can be positioned so as to abut the outer peripheral surface 332 of the cell culture surface 315. For example, in some embodiments, the opening 323a of the microcavity 320a is such that the cells suspended in the liquid do not settle or adhere to any other surface of the container 300. It may be coplanar with the outer surface 332 of the cell culture surface 315 as it falls into the well 322a of 320a (eg, at least based on gravity) and / or is directed by the outer surface 332. In an embodiment, the cell culture surface 315 extends from wall 301 to wall 301. In embodiments, the cell culture surface 315 does not have any flat area. That is, the cell culture surface is an array of microcavities 320 extending from wall to wall with no edges or flat areas between the cell culture surface and the wall 301. In embodiments, the cell culture surface is substantially composed of multiple microcavities. In embodiments, there is no flat area in the cell culture chamber where the cells settle. This is important to ensure that the cells do not colonize the cell culture chamber outside the microwell. When cells colonize a flat area outside the cell culture surface outside the microwell, the cells proliferate as irregular cell aggregates 801 (see Figures 35A and 35B, 36A and 36B) and are multicellular in their vessels. It can form heterogeneous populations of 3D structures. In embodiments, the cell culture surface is substantially composed of multiple microcavities.

In some embodiments, cells that settle or adhere to the surface of the container 300 accumulate and grow (eg, proliferate) outside the microcavities 320a, 320b, 320c, and the microcavities 320a, 320b, Problems can arise with respect to the desired proliferation of 3D cells within the 320c. 35A and 35B are schematic views of cells accumulating in a flat area around the outer periphery of a microcavity. For example, in some embodiments, cells that do not fall into wells 322a, 322b, 222c (at least based on gravity) and accumulate or adhere to other surfaces of container 300 are wells 322a, 322b, 222c. Can grow outside of. When cells colonize a flat area outside the cell culture surface outside the microwells, the cells can proliferate as unwanted irregular cell aggregates 801. In addition, these irregular cell aggregates 801 enter into adjacent microcavities and disrupt the desired growth of three-dimensional cells within wells 322a, 322b, and 222c (eg, block, alter, and slow). Or hinder) get. Similarly, in some embodiments, cells that accumulate or adhere to the other surface of the vessel 300 proliferate and displace the three-dimensional cells within wells 322a, 322b, and 322c, thereby pushing wells 322a, 322b. It can disrupt or impair the desired proliferation of 3D cells within 322c and alter the desired size uniformity of the cells. Therefore, in some embodiments, by locating at least a portion 331 of the outer circumference 330 of the cell culture surface 315 in the recess 335 or on the protrusion 337, all cells suspended in the liquid are placed in the well 322a. It can reduce or eliminate problems that would otherwise occur if cells adhere to the surface of the outer vessel 300 of wells 322a, 322b, and 322c towards the inside of, 322b, 322c.

Another exemplary embodiment of the cell culture vessel 300 is shown in the cross section of FIG. In some embodiments, the cell culture vessel 300 and the cell culture surface 315 can be made from the same material. For example, in some embodiments, the cell culture surface 315 with the plurality of microcavities 320 is manufactured as an integral part of the wall 301 of the container 300 (eg, shaping, machining, pressing, extrusion, molding, 3D printing). It can be printed, etc.), so there is no direct boundary between the cell culture surface 315 and the wall 301 of the container 300. As shown in FIG. 30, in some embodiments, the cell culture surface 315 and the inner surface 302 of the wall 301 (both integrally formed) can define the cell culture chamber 303 of the container 300. .. Alternatively, as shown in FIG. 31, in some embodiments, the inner surface 302 of the wall 301 defines the cell culture chamber 303 of the container 300, including a first region 303a and a second region 303b. The cell culture surface 315 (formed integrally with the wall 301) can be positioned within the cell culture chamber 303 between the first region 303a and the second region 303b. In some embodiments, the container 300 containing the wall 301 and the cell culture surface 315, both integrally manufactured, may contain a material that is impermeable. Alternatively, in some embodiments (eg, when the cell culture surface 315 is attached to the wall 301 of the container 300), the wall 301 of the container 300 can be made from a non-permeable material and the cell culture. The surface 315 can be made from one or more of a non-permeable material, a non-porous material, a gas permeable material, or a porous material formed integrally with the wall 301.

In this embodiment, where the cell culture surface 315 and wall 301 are manufactured as a single component, the cell culture surface 315 extends from wall 301 to wall 301. In embodiments, the cell culture surface 315 does not have any flat area. That is, the cell culture surface is an array of microcavities 320 extending from wall to wall with no edges or flat areas between the cell culture surface and the wall 301. In embodiments, the cell culture surface is substantially composed of multiple microcavities. In embodiments, there is no flat area in the cell culture chamber where the cells settle. This is important to ensure that the cells do not colonize the cell culture chamber outside the microwell. When cells colonize a flat area outside the cell culture surface outside the microwell, the cells proliferate as irregular cell aggregates 801 (see Figures 35A and 35B, 36A and 36B) and are multicellular in their vessels. It can form heterogeneous populations of 3D structures. In embodiments, the cell culture surface is substantially composed of multiple microcavities.

Therefore, in some embodiments, the container 300 can contain a predetermined amount of liquid 370, and the method of culturing cells in the cell culture container 300 is a method of culturing cells in at least one microcavity 320a of a plurality of microcavities 320. , 320b, 320c, and after depositing the liquid 370 in at least one microcavity 320a, 320b, 320c, the cells are cultured in at least one microcavity 320a, 320b, 320c. Can include steps.

As shown in FIG. 32, which shows an enlarged view of the cell culture surface 315 and the wall 301 integrally formed with the container 300 in the area 32 of FIG. 30, a predetermined amount of liquid 370 and the submerged surface 325 of the container 300. It can be contacted and occupy some area of the cell culture chamber 303 of the container 300. FIG. 33 shows an alternative exemplary embodiment of FIG. 32, including features of at least a portion 331 of the outer circumference 330 of the cell culture surface 315 located within the recess 335 of the wall 301 of the container 300. A predetermined amount of liquid comes into contact with the submerged surface 325 of the container 300, including the outer peripheral surface 332 of the cell culture surface 315, and occupies a region of the cell culture chamber 303 of the container 300. Similarly, FIG. 34 shows an alternative exemplary embodiment of FIG. 32, including features of at least a portion 331 of the outer circumference 330 of the cell culture surface 315 located on the protrusion 337 of the wall 301 of the container 300. The predetermined amount of liquid 370 is in contact with the submerged surface 325 of the container 300, including the outer peripheral surface 332 of the cell culture surface 315, and occupies a certain area of the cell culture chamber 303 of the container 300.

For explanatory purposes only, in some embodiments, the submerged surface 325 may include the surface of the container 300 in contact with a predetermined amount of liquid 370, FIGS. 32-34. The thickness of the extra-thick line in is indicated by the submerged surface 325. In some embodiments, the submerged surface 325 can be defined for the direction of gravity "g" at or during a particular step of the method of culturing cells in a container 300. For example, in some embodiments, a predetermined amount of liquid 370 can define a liquid level 371, where the submerged surface 325 is a liquid level 371 in the direction of gravity "g". Includes the surface of a container 300 that is located below and is therefore in contact with a predetermined amount of liquid 370, submerged in a predetermined amount of liquid 370. In some embodiments, the liquid level 371 of the predetermined amount of liquid 370 defines a flat free surface of the predetermined amount of liquid 370 spaced a distance from a portion 375 of the cell culture surface 315. Can be done. For example, a portion 375 of the cell culture surface 315 can include openings 323a, 323b, 323c, and a flat free surface defined by the liquid level 371 of a predetermined amount of liquid 370 is each of the plurality of microcavities 320. The concave surfaces 321a, 321b, 321c of the microcavities 320a, 320b, 320c can be separated from a part of 375 by a certain distance in the direction away from the concave surfaces 321a, 321b, 321c.

In addition, in some embodiments, the submerged surface 325 of the container 300 does not include a flat surface portion parallel to the flat free surface of a predetermined amount of liquid 370. By providing a submerged surface 325 that does not include a flat surface portion parallel to the flat free surface of the liquid surface 371 of a predetermined amount of liquid 370, the cells suspended in the liquid 370 can be colonized. Or, because there is no submerged surface 325 to which cells can adhere, they fall into wells 322a, 322b, 322c of the microcavities 320a, 320b, 320c (eg, at least based on gravity) and / or are directed by the submerged surface 325. Be done. As mentioned above, in some embodiments, cells that settle or adhere to the surface of the container 300 accumulate and grow (eg, proliferate) outside the microcavities 320a, 320b, 320c and microcavities. Problems can arise with respect to the desired proliferation of three-dimensional cells within 320a, 320b, 320c. For example, in some embodiments, cells that do not fall into wells 322a, 322b, 222c (at least based on gravity) and accumulate or adhere to other surfaces of the vessel 300 (eg, submerged surface 325). Can grow outside wells 322a, 322b, and 322c, and three-dimensional cells within wells 322a, 322b, and 322c. Can disrupt the desired growth of (eg, block, alter, slow or hinder). Similarly, in some embodiments, if the submerged surface 325 contains a flat surface portion parallel to the flat free surface of a predetermined amount of liquid 370, the cells are on the flat surface portion. It can accumulate or adhere and proliferate, pushing away the 3D cells in wells 322a, 322b, and 222c, thereby disturbing or impairing the desired growth of 3D cells in wells 322a, 322b, 222c. Therefore, in some embodiments, a submerged surface 325 that does not include a flat surface portion parallel to the flat free surface of the liquid surface 371 of a predetermined amount of liquid 370 is provided to suspend in the liquid 370. All turbid cells are directed into wells 322a, 322b, and 222c, thus reducing possible problems if cells adhere to the surface of the outer vessel 300 of wells 322a, 322b, 222c. Can be made or eliminated.

As shown in FIG. 33, in some embodiments, at least a portion 331 of the outer circumference 330 of the cell culture surface 315 is the concave surfaces 321a, 321b of the respective microcavities 320a, 320b, 320c of the plurality of microcavities 320. A distance "d4" can be provided from a part 375 of the cell culture surface 315 in the direction away from 321c. In addition, the cell culture surface 315 can include an outer peripheral surface 332 extending from at least a portion 331 of the outer circumference 330 to a portion 375 of the cell culture surface 315. In some embodiments, the depth "d5" of a predetermined amount of liquid 370 from the liquid level 371 defining a flat free surface along the direction to a portion 375 of the cell culture surface 315 is a distance. It can be smaller than "d4". Similarly, as shown in FIG. 34, in some embodiments, at least a portion 331 of the outer circumference 330 of the cell culture surface 315 is a concave surface 321a of each of the microcavities 320a, 320b, 320c of the plurality of microcavities 320. , 321b, 321c can be separated from a part 375 of the cell culture surface 315 by a distance "d6". The cell culture surface 315 can include an outer peripheral surface 332 extending from at least a portion 331 of the outer circumference 330 to a portion 375 of the cell culture surface 315. In some embodiments, the depth "d7" of a predetermined amount of liquid 370 from the liquid level 371 defining a flat free surface along the direction to a portion 375 of the cell culture surface 315 is a distance. It can be smaller than "d6".

Thus, in some embodiments, alone or in combination, a submerged surface 325 that does not include a flat surface portion parallel to the flat free surface of the liquid surface 371 of a predetermined amount of liquid 370 is provided. The depth "d5" of a predetermined amount of liquid 370 from the liquid surface 371 defining a flat free surface along the direction to a portion 375 of the cell culture surface 315 is made smaller than the distance "d4". The depth of a predetermined amount of liquid 370 can be (eg, including recess 335, FIG. 33), from the liquid level 371 defining a flat free surface along the direction to a portion 375 of the cell culture surface 315. The "d7" can be less than the distance "d6" (eg, including protrusions 337, FIG. 34), and all cells suspended in liquid 370 are placed in wells 322a, 322b, and 322c. Orientation and therefore can reduce or eliminate problems that would otherwise occur if cells adhere to the surface of the outer vessel 300 of wells 322a, 322b, 322c. Furthermore, although not explicitly shown, in some embodiments, the method of culturing cells (see FIG. 16) in a container 300 is at least one of a plurality of microcavities 320a, 320b, 320c. , A step of depositing a portion of a predetermined amount of liquid 370 in 320b, 320c, and after depositing a portion of a predetermined amount of liquid 370 in at least one microcavity 320a, 320b, 320c, at least one micro. A step of culturing cells in the cavities 320a, 320b, 320c can be included.

Referring to FIGS. 32 to 34, in some embodiments, the method of culturing cells in the cell culture vessel 300 involves filling a region of the cell culture chamber 303 of the vessel 300 with a predetermined amount of liquid 370. Can include. In some embodiments, the cell culture chamber 303 can be defined at least partially by the inner surface 302 of the wall 301 of the container 300, the method of which is at least a plurality of microcavities 320 of the cell culture surface 315. A step of depositing a part of a predetermined amount of liquid 370 in one microcavity 320a, 320b, 320c can be included. The cell culture surface 315 can define at least a part 331 of the region, and each of the microcavities 320a, 320b, 320c of the plurality of microcavities 320 has concave surfaces 321a, 321b that define wells 322a, 322b, and 322c. , 321c and openings 323a within a portion 375 of cell culture surface 315 defining passages to wells 322a, 322b, and 322c. In some embodiments, the method involves depositing a portion of a predetermined amount of liquid 370 in at least one microcavity 320a, 320b, 320c and then submerging the surface of the container 300 with the predetermined amount of liquid 370. A step of culturing cells in at least one microcavity 320a, 320b, 320c while in contact with 325 can be further included. In some embodiments, while culturing cells in at least one microcavity 320a, 320b, 320c, the submerged surface 325 is a flat surface containing a surface normal opposite the direction of gravity "g". Does not include parts. If desired, in some embodiments, while culturing cells in at least one microcavity 320a, 320b, 320c of the plurality of microcavities 320, the liquid level 371 of a predetermined amount of liquid 370 is gravitational. A flat free surface of a predetermined amount of liquid 370, perpendicular to the "g" direction, can be defined.

By providing a submerged surface 325 that does not contain a flat surface with a surface normal opposite the direction of gravity "g", cells suspended in liquid 370 can be colonized or adhered to. Since there is no submerged surface 325 that can be, it is directed by the submerged surface 325 that falls (eg, at least based on gravity) into the wells 322a, 322b, and 322c of the microcavities 320a, 320b, 320c. As mentioned above, in some embodiments, cells that settle or adhere to the surface of the container 300 accumulate and grow (eg, proliferate) outside the microcavities 320a, 320b, 320c and microcavities. Problems can arise with respect to the desired proliferation of three-dimensional cells within 320a, 320b, 320c. For example, in some embodiments, cells that do not fall into wells 322a, 322b, 222c (at least based on gravity) and accumulate or adhere to other surfaces of the vessel 300 (eg, submerged surface 325). Can proliferate outside wells 322a, 322b, and 322c, and of three-dimensional cells within wells 322a, 322b, and 322c), including a flat surface portion containing a surface normal opposite the direction of gravity "g". It can disrupt the desired growth (eg, block, modify, slow or hinder). Similarly, in some embodiments, if the submerged surface 325 contains a flat surface portion containing a surface normal opposite to the direction of gravity "g", the cells accumulate on that flat surface portion. Alternatively, they can adhere and proliferate, pushing away the 3D cells in wells 322a, 322b, and 222c, thereby disturbing or impairing the desired proliferation of 3D cells in wells 322a, 322b, and 222c. Thus, in some embodiments, all suspended in liquid 370 by providing a submerged surface 325 that does not include a flat surface portion that includes a surface normal opposite the direction of gravity "g". Direct the cells into wells 322a, 322b, 222c and therefore reduce or reduce problems that may otherwise occur if the cells colonize or adhere to the surface of the outer vessel 300 of wells 322a, 322b, 322c. Can be eliminated.

Further, for the purposes of the present disclosure, unless otherwise stated, "flat surface portion" is intended to mean any flat surface portion, including planar dimensions greater than about 5 micrometers. For example, in some embodiments, a submerged surface 325 that does not include a flat surface portion parallel to the flat free surface of the liquid level 371 of a given amount of liquid 370 is the liquid level of the given amount of liquid 370. It can be defined as a submerged surface 325 that is parallel to the flat free surface of 371 and does not include a flat surface portion with a planar dimension of more than about 5 micrometer. Similarly, in some embodiments, a submerged surface 325 that does not include a flat surface portion that includes a surface normal opposite the direction of gravity "g" is a surface normal opposite the direction of gravity "g". Can be defined as a submerged surface 325 that does not include a flat surface portion that includes a planar dimension greater than about 5 micrometers.

For example, in some embodiments, the submerged surface 325 may include a planar portion; however, if the planar dimension of the flat surface portion is, for example, 5 micrometers or less, in some embodiments. In, the flat surface portion is considered to be too small for cells to reasonably accumulate or adhere. Thus, in some embodiments, all cells suspended in liquid 370 are well-welled by providing a submerged surface 325 that does not include a flat surface portion that includes a planar dimension greater than about 5 micrometers. Oriented into 322a, 322b, 322c and therefore can reduce or eliminate problems that would otherwise occur if cells colonize or adhere to the surface of the outer vessel 300 of wells 322a, 322b, 322c. .. However, in some embodiments, the submerged surface 325 can completely eliminate the flat surface portion, regardless of the threshold dimensions that define the flat surface portion.

35A and 35B are schematics showing cells proliferating as spheroids and cells proliferating as irregular cell aggregates 801 in the microcavity. These irregular cell aggregates are the embodiments shown in FIGS. 1-16 (cell culture surface 115) or the embodiments shown in FIGS. 17-34, where a flat surface occurs in the vessel. It can occur at (cell culture surface 315). In order to avoid these irregular cell aggregates, it is important to avoid flat surfaces within the cell culture chamber 103 or 303.

FIG. 36A is a photograph of spheroids in an array of microcavities under suitable conditions to provide a homogeneous population of spheroids in a container. FIG. 36B is a photograph of an irregular cell assembly 801 isolated from a container having a flat surface within cell culture chambers 103, 303. Embodiment of a cell culture vessel having no flat surface on the cell culture surface or no flat surface in the submerged area of the cell culture surface in order to avoid the production of these irregular cell aggregates 801 Is provided. That is, in an embodiment of a cell culture vessel, the cell culture surface becomes substantially from an array of microcavities and does not provide a flat surface capable of producing unwanted irregular cell aggregates 801.

Throughout the disclosure, the terms "material," "liquid," and "gas" may be used, for example, to describe the properties of the material used when culturing cells in a cell culture vessel. it can. Unless otherwise stated, for the purposes of the present disclosure, a "material" may include a fluid material (eg, liquid or gas). In addition, the material can include a culture medium or medium containing a liquid containing solid particles (eg, cells) suspended in the liquid. Unless otherwise stated, for the purposes of the present disclosure, a "liquid" is used, for example, to clean a cell culture chamber, to kill one or more characteristic structures of a cell culture surface and container, to allow cell growth and liquid It can contain a wash or rinse solution, aqueous solution, or other liquid that can be added to or removed from the container to prepare the cell culture surface for other uses. In addition, the liquid can include a culture medium or medium containing the liquid containing solid particles (eg, cells) suspended in the liquid. Unless otherwise stated, for the purposes of the present disclosure, "gas" may include air, filtered or treated air, or other gases.

Throughout the present disclosure, the terms "impermeable", "gas permeable", and "porous" are the properties of one or more feature structures of the cell culture surface (eg, material properties, features, parameters). Can be used to describe.

Unless otherwise stated, for the purposes of the present disclosure, "impermeable" cell culture surfaces (eg, materials for non-permeable cell culture surfaces) are in standard state (eg, but not limited to, pressure and). It is considered impermeable to solids, liquids, and gases (without external influences, including force) and is therefore impervious to, through, or in its impermeable cell culture surface under standard conditions. Does not allow the movement of solids, liquids, or gases out of. In some embodiments, the impermeable cell culture surface can form part of the wall of the vessel. In addition, the cell culture chamber of the container is sterile if the impermeable cell culture surface forms part of the wall of the container, for example, because bacteria cannot pass through the impermeable cell culture surface. Conceivable. However, when multiple microcavities on the cell culture surface are filled with material, the gas is trapped in the microcavities on the impermeable cell culture surface based on the surface tension of the liquid, thereby causing some. In embodiments, it can prevent the material from filling the microcavities and prevent the growth of spheroids.

Unless otherwise stated, for the purposes of the present disclosure, "gas permeable" cell culture surfaces (eg, gas permeable cell culture surface materials) are impervious to solids and liquids under standard conditions. , Is considered to be permeable to gas. Thus, the gas permeable cell culture surface does not allow the movement of solids and liquids into and out of the gas permeable cell culture surface, and into the gas permeable cell culture surface. Allows the movement of gas through or out of it. In some embodiments, the gas permeable cell culture surface can form part of the wall of the vessel. In addition, the cell culture chamber of the container is to the extent that the gas permeable cell culture surface forms part of the wall of the container, for example, bacteria are not perfect but satisfactory on the gas permeable cell culture surface. It is considered sterile because it cannot pass through. However, although the cell culture surface is gas permeable, the gas permeation rate through the gas permeable cell culture surface is slower than the rate required to move the gas out of the microcavity under normal operating conditions. It is possible and therefore gas can still be trapped in the microcavities during material filling, as it can take an unacceptably long time to permeate through the cell culture surface. Therefore, in some embodiments, slow filling of the microcavities can cause the front surface of the liquid to enter each microcavity at an angle, thereby moving the gas as the liquid fills the microcavities. it can. In some embodiments, after filling the microcavities with a liquid, the gas can permeate (slowly) through the surface of the gas permeable cell culture.

Unless otherwise stated, for the purposes of the present disclosure, "porous" cell culture surfaces (eg, materials for porous cell culture surfaces) are impermeable to solids under standard conditions, liquids and gases. Is considered to be transparent to. Thus, the porous cell culture surface does not allow the movement of solids into or out of the porous cell culture surface, and into or through the porous cell culture surface. Allows the movement of liquids and gases out of it. The porous cell culture surface cannot form part of the vessel because bacteria can pass through the porous cell culture surface and therefore can cause sterility problems in the cell culture chamber. Therefore, when using a porous cell culture surface, the cell culture surface must be sealed (completely sealed) within the sterile cell culture chamber of the container. However, during filling of the microcavities with material, the gas can leak (eg, pass) through the surface of the porous cell culture. Therefore, filling of the microcavity can be performed quickly without fear of trapping gas in the microcavity. In some embodiments, the liquid can pass through the porous cell culture surface only by increasing pressure or physical contact and disturbance of the cell culture surface. Therefore, in some embodiments, the material containing the liquid is housed within the microcavities of the cell culture surface unless the cell culture surface is exposed to increased pressure or physical contact and disturbance of the cell culture surface. Can be done. For example, in some embodiments, a porous cell culture surface is supported in a cell culture chamber and gas is allowed to pass through the cell culture surface during filling and culture, and the cell culture surface is subjected to pressure. It can be isolated from increased or disturbances from physical contact and external forces (eg, outside the cell culture chamber).

Numerous aspects of cell culture vessels and methods of culturing cells have been disclosed herein. A summary of some selected embodiments is presented below.

In a first aspect, the present disclosure is a cell culture vessel comprising a cell culture surface substantially consisting of a plurality of microcavities; a wall attached to the cell culture surface; the cell culture surface and the cell culture surface thereof. Provided is a cell culture vessel in which the inner surface of the wall defines the cell culture chamber of this vessel.

In a second aspect, the present disclosure provides the cell culture vessel of aspect 1, wherein each microcavity of the plurality of microcavities comprises a concave bottom and an opening.

In a third aspect, the disclosure provides a cell culture vessel of aspect 1 or 2, further comprising a necked opening.

In a fourth aspect, the disclosure provides a cell culture vessel of aspect 3, further comprising a dam at its necked opening.

In a fifth aspect, the disclosure provides a cell culture vessel of aspect 1 or 2, further comprising a lid.

In a sixth aspect, the present disclosure provides a cell culture vessel of aspect 3 or 4, further comprising a lid.

In a seventh aspect, the present disclosure provides the cell culture vessel of aspect 6, wherein the lid comprises a hinged opening.

In an eighth aspect, the present disclosure provides a cell culture vessel of aspect 4, wherein the lid comprises a sliding opening.

In a ninth aspect, the present disclosure provides a cell culture vessel of aspects 3, 4, or 6-8, wherein the necked opening comprises a bend.

In a tenth aspect, the disclosure provides a cell culture vessel according to any one of aspects 1-9, wherein the wall comprises a recess and the cell culture surface is attached to the recess.

In an eleventh aspect, the disclosure provides a cell culture vessel according to any one of aspects 1-9, wherein the wall comprises a protrusion and the cell culture surface is attached to the protrusion.

In a twelfth aspect, the present disclosure comprises a cell culture vessel comprising a cell culture surface comprising a plurality of microcavities having a non-flat sinusoidal shape; a wall attached to the cell culture surface thereof. A cell culture vessel is provided in which the cell culture surface and the inner surface of the wall define the cell culture chamber of the container, and the cell culture surface substantially does not contain a flat area.

In a thirteenth aspect, the present disclosure provides a cell culture vessel of aspect 12, wherein each microcavity of the plurality of microcavities comprises a concave bottom and an opening.

In a fourteenth aspect, the present disclosure provides a cell culture vessel of aspect 12 or 13, further comprising a necked opening.

In a fifteenth aspect, the disclosure provides a cell culture vessel of aspect 14, further comprising a dam at its necked opening.

In a sixteenth aspect, the disclosure provides a cell culture vessel of aspect 12 or 13, further comprising a lid.

In a seventeenth aspect, the disclosure provides a cell culture vessel of aspect 14 or 15, further comprising a lid.

In an eighteenth aspect, the present disclosure provides a cell culture vessel of aspect 17, wherein the lid comprises a hinged opening.

In a nineteenth aspect, the present disclosure provides a cell culture vessel of aspect 17, wherein the lid comprises a sliding opening.

In a twentieth aspect, the present disclosure provides a cell culture vessel of aspects 14, 15, or 17-19, wherein the necked opening comprises a bend.

In a twenty-first aspect, the present disclosure provides a 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.

In a twenty-second aspect, the present disclosure provides a cell culture vessel of any one of aspects 12-17, wherein the wall comprises a protrusion and the cell culture surface is attached to the protrusion.

It will be appreciated that the various disclosed embodiments may include specific feature structures, elements or steps described for that particular embodiment. A particular feature structure, element or process is described in relation to one particular embodiment, but may be replaced with an alternative embodiment in various combinations or sequences not shown, or It will also be recognized that they can be combined.

As used herein, it should be understood that nouns refer to "at least one" object and should not be limited to "only one" unless otherwise stated. Thus, for example, reference to "ingredients" includes embodiments having two or more such ingredients, unless otherwise stated.

The range can be expressed herein from "about" one particular value and / or "about" another particular value. When such a range is expressed, embodiments include from that one particular value and / or to the other particular value. Similarly, it will be understood that when a value is expressed as an approximation using the antecedent "about", that particular value forms another aspect. It will be further understood that each endpoint of the range is significant both in relation to the other endpoint and regardless of the other endpoint.

Unless otherwise stated, none of the methods described herein is intended to be construed as requiring the steps to be performed in a particular order. Therefore, it is otherwise specifically stated in the claims or description that the claims of the method do not actually enumerate the order in which the steps should be followed, or that the steps are limited to a particular order. If not, it is never intended to imply any particular order.

Various feature structures, elements or steps of a particular embodiment can be disclosed using the transition phrase "contains", but using the transition phrase "consisting of" or "consisting of substantially". It should be understood that alternative embodiments, including those that can be described, are implied. Thus, for example, alternative embodiments implied for a device comprising A + B + C include embodiments in which the device comprises A + B + C and embodiments in which the device substantially comprises A + B + C.

It will be apparent to those skilled in the art that various modifications and changes to this disclosure can be made without departing from the spirit and scope of this disclosure. Therefore, the present disclosure is intended to include modifications and modifications of the present disclosure on the premise that they fall within the scope of the accompanying claims and their equivalents.

Hereinafter, preferred embodiments of the present invention will be described in terms of terms.

Embodiment 1
It is a cell culture container
With a cell culture surface that is substantially composed of multiple microcavities,
The wall attached to the cell culture surface and
With
A cell culture container in which the cell culture surface and the inner surface of the wall define the cell culture chamber of the container.

Embodiment 2
The cell culture vessel according to embodiment 1, wherein each microcavity of the plurality of microcavities includes a concave bottom and an opening.

Embodiment 3
The cell culture vessel according to embodiment 1 or 2, further comprising a necked opening.

Embodiment 4
The cell culture vessel according to embodiment 3, further comprising a dam in the necked opening.

Embodiment 5
The cell culture vessel according to embodiment 1 or 2, further comprising a lid.

Embodiment 6
The cell culture vessel according to embodiment 3 or 4, further comprising a lid.

Embodiment 7
The cell culture vessel according to embodiment 6, wherein the lid includes a hinged opening.

8th Embodiment
The cell culture vessel according to embodiment 4, wherein the lid includes a sliding opening.

Embodiment 9
The cell culture vessel according to any one of Embodiments 3, 4, and 6 to 8, wherein the necked opening includes a bent portion.

Embodiment 10
The cell culture container according to any one of embodiments 1 to 9, wherein the wall includes a recess and the cell culture surface is attached to the recess.

Embodiment 11
The cell culture container according to any one of embodiments 1 to 9, wherein the wall includes a protrusion and the cell culture surface is attached to the protrusion.

Embodiment 12
It is a cell culture container
A cell culture surface containing multiple microcavities with a non-flat sinusoidal shape,
The wall attached to the cell culture surface and
With
The cell culture surface and the inner surface of the wall define the cell culture chamber of this container.
A cell culture vessel that substantially does not contain an area where the cell culture surface is flat.

Embodiment 13
The cell culture vessel according to embodiment 12, wherein each microcavity of the plurality of microcavities includes a concave bottom and an opening.

Embodiment 14
The cell culture vessel according to embodiment 12 or 13, further comprising a necked opening.

Embodiment 15
The cell culture vessel according to embodiment 14, further comprising a dam in the necked opening.

Embodiment 16
The cell culture vessel according to embodiment 12 or 13, further comprising a lid.

Embodiment 17
The cell culture vessel according to embodiment 14 or 15, further comprising a lid.

Embodiment 18
The cell culture vessel according to embodiment 17, wherein the lid comprises a hinged opening.

Embodiment 19
The cell culture vessel according to embodiment 17, wherein the lid comprises a sliding opening.

20th embodiment
The cell culture vessel according to any one of embodiments 14, 15, and 17 to 19, wherein the necked opening comprises a bend.

21st embodiment
The cell culture vessel according to any one of embodiments 12 to 17, wherein the wall includes a recess and the cell culture surface is attached to the recess.

Embodiment 22
The cell culture vessel according to any one of embodiments 12 to 17, wherein the wall includes a protrusion and the cell culture surface is attached to the protrusion.

100, 300 Cell culture vessel 101 Top 103, 303 Cell culture chamber 104 Cap 105 Opening 107, 301 Wall 108 Bottom 112, 507 Opening 115, 315 Cell culture surface 120, 120a, 120b, 120c, 320, 320a, 320b, 320c Microcavities 121a, 121b, 121c, 321a, 321b, 321c Concave surfaces 122a, 122b, 122c, 322a, 322b, 322c Wells 123a, 123b, 123c, 323a, 323b, 323c Openings 130 Dam 136 Openings 137, 304 Lid 140, 370 Predetermined amount of liquid 150, 150a, 150b, 150c, 350 cells, spheroid 158 Bending part 180 Groove 182 Hinge 200 Cell culture surface 330 Overall circumference 335 Recessed part 337 Projection part 555, 555a Neck part 801 Multicellular 3D aggregate

Claims (22)

It is a cell culture container
With a cell culture surface that is substantially composed of multiple microcavities,
The wall attached to the cell culture surface and
With
A cell culture container in which the cell culture surface and the inner surface of the wall define the cell culture chamber of the container. The cell culture vessel according to claim 1, wherein each microcavity of the plurality of microcavities includes a concave bottom and an opening. The cell culture vessel according to claim 1 or 2, further comprising a necked opening. The cell culture vessel according to claim 3, further comprising a dam in the necked opening. The cell culture vessel according to claim 1 or 2, further comprising a lid. The cell culture vessel according to claim 3 or 4, further comprising a lid. The cell culture vessel according to claim 6, wherein the lid includes a hinged opening. The cell culture vessel according to claim 4, wherein the lid includes a sliding opening. The cell culture vessel according to any one of claims 3, 4, and 6 to 8, wherein the necked opening includes a bent portion. The cell culture container according to any one of claims 1 to 9, wherein the wall includes a recess and the cell culture surface is attached to the recess. The cell culture container according to any one of claims 1 to 9, wherein the wall includes a protrusion and the cell culture surface is attached to the protrusion. It is a cell culture container
A cell culture surface containing multiple microcavities with a non-flat sinusoidal shape,
The wall attached to the cell culture surface and
With
The cell culture surface and the inner surface of the wall define the cell culture chamber of this container.
A cell culture vessel that substantially does not contain an area where the cell culture surface is flat. 12. The cell culture vessel according to claim 12, wherein each microcavity of the plurality of microcavities includes a concave bottom and an opening. The cell culture vessel according to claim 12 or 13, further comprising a necked opening. The cell culture vessel according to claim 14, further comprising a dam in the necked opening. The cell culture vessel according to claim 12 or 13, further comprising a lid. The cell culture vessel according to claim 14 or 15, further comprising a lid. The cell culture vessel according to claim 17, wherein the lid includes a hinged opening. The cell culture vessel according to claim 17, wherein the lid includes a sliding opening. The cell culture vessel according to any one of claims 14, 15, and 17 to 19, wherein the necked opening includes a bent portion. The cell culture container according to any one of claims 12 to 17, wherein the wall includes a recess and the cell culture surface is attached to the recess. The cell culture container according to any one of claims 12 to 17, wherein the wall includes a protrusion and the cell culture surface is attached to the protrusion.

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