Cell culture apparatus and method for using the same

Abstract

This disclosure describes cell culture apparatus having non-standard wall structures and / or shapes. The cell culture apparatus described herein may include terraced or stepped wall segments, where consecutive terraces or steps are dimensionally wider. This disclosure also relates to the use of the described cell culture apparatus in a method or process.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 457,583, filed on April 6, 2023, the entire content of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to cell culture, more specifically to an apparatus for cell culture, and even more specifically to uses or methods of using such an apparatus for cell culture.

Background Art

[0003] In vitro or ex vivo cell culture systems increasingly rely on culturing cells within or on an extracellular matrix because the extracellular matrix provides support and / or signals that mimic the in vivo environment. In particular, current organoids and other 3D models tend to rely on support from an extracellular matrix or mimetic.

[0004] Organoids and other three - dimensional models are becoming increasingly widespread in the cell culture workflow because they represent a more physiologically relevant model system in terms of tissue / organ structure and cellular composition compared to 2D adherent cultures and non - adherent cultures. In fact, organoids and other three - dimensional model systems can enable a more accurate response to drugs and other compounds and may simplify the path to the clinic. Furthermore, as more physiologically relevant model systems, organoids and other three - dimensional models may be more suitable for applications in cell therapy and regenerative medicine.

[0005] The improvement in the physiological relevance of 3D model systems can be achieved by better supporting biological complexity, but such 3D systems often come with increased heterogeneity. On the one hand, heterogeneity may arise for reasons inherent to the biological system, which in itself may be why organoid systems better reproduce physiological relevance compared to conventional systems that support less diverse cell types or lack an appropriate spatial configuration of cell types. On the other hand, 3D approaches are usually technically challenging and can lead to experimental heterogeneity.

[0006] The current state of organoid or three-dimensional tissue generation involves forming a dome of extracellular matrix containing cells on the surface of a conventional flat-bottomed culture dish or plate. Such dome cultures have many drawbacks, including variations in mass transfer rates depending on the localization of cells within the dome, and the inability to control the shape or surface position of the dome.

[0007] Therefore, there is a need for improved cell culture systems that enable the biological complexity of three-dimensional culture models while mitigating the associated technical shortcomings and challenges. [Overview of the Initiative]

[0008] This disclosure relates to cell culture, and more specifically to apparatus for cell culture, and uses or methods of using such apparatus for cell culture. In certain embodiments, apparatus for culturing cells is described. In certain embodiments, methods or processes of cell culture using the apparatus of this disclosure are described.

[0009] In one aspect of the present disclosure, a cell culture apparatus is provided. The cell culture apparatus of the present disclosure may include a well having a bottom wall and one or more side walls defining an opening opposite the bottom wall. In one embodiment, the cell culture apparatus includes a plurality of side wall segments having a continuous circumference.

[0010] In one embodiment, the cell culture apparatus (more specifically, its wells) includes a first sidewall segment connected to the bottom wall and extending away from the bottom wall. The first sidewall segment and the bottom wall define a first volume boundary capable of holding or storing liquid and / or polymerizable liquid.

[0011] The cell culture apparatus (more specifically its wells) may further include a second sidewall segment connected to a first sidewall segment by a first ledge. The second sidewall segment defines the boundary of the space above the first volume, and thereby the planes of the second sidewall segment and the first ledge define the boundary of a second volume that can hold or store a liquid and / or polymerizable liquid (provided that the first volume is filled with a liquid or polymerizable liquid).

[0012] In one embodiment, the height of the first side wall segment is greater than the height of the second side wall segment.

[0013] In one embodiment, the first volume is equal to the second volume with an error of ±10%. In one embodiment, the first volume and the second volume are substantially equal or equal.

[0014] In one embodiment, the height of the first side wall segment is approximately 0.1 mm to 10 mm, preferably 0.5 mm to 5 mm. In the same or a different embodiment, the height of the second side wall segment is approximately 0.1 mm to 10 mm, preferably 0.2 mm to 4 mm. In one embodiment, the height of the first side wall segment is greater than the height of the second side wall segment.

[0015] In one embodiment, the first sidewall segment forms an obtuse angle with the bottom wall, and / or the second sidewall segment forms an obtuse angle with the first ledge. In one embodiment, the angle is 90 to 100 degrees.

[0016] In one embodiment, the width of the first ledge is approximately 0.1 mm to approximately 5 mm. In another embodiment, the width of the first ledge is approximately 1 to 2 mm ± 0.25 mm.

[0017] In one embodiment, one or both of the first and second sidewall segments are substantially circular in the plane of the bottom wall (for example, when viewed from the opening toward the bottom wall). In one embodiment, the diameter of the first sidewall segment at any point along its height is smaller than the diameter of the second sidewall segment at any point along its height. The diameters of the first and second sidewall segments vary depending on the form of the plate.

[0018] In one embodiment, the diameter of the first sidewall segment (along the widest plane) is approximately 2 mm to 35 mm. In one embodiment, the diameter of the first sidewall segment (along the widest plane) is approximately 2 mm to 30 mm. In one embodiment, the diameter of the first sidewall segment (along the widest plane) is approximately 2 mm to 25 mm. In one embodiment, the diameter of the first sidewall segment (along the widest plane) is approximately 2 mm to 20 mm. In one embodiment, the diameter of the first sidewall segment (along the widest plane) is approximately 2 mm to 15 mm. In one embodiment, the diameter of the first sidewall segment (along the widest plane) is approximately 2 mm to 10 mm.

[0019] In one embodiment, the diameter of the second sidewall segment (along the widest plane) is approximately 1 mm to 40 mm. In one embodiment, the diameter of the second sidewall segment (along the widest plane) is approximately 2 mm to 35 mm. In one embodiment, the diameter of the second sidewall segment (along the widest plane) is approximately 2 mm to 30 mm. In one embodiment, the diameter of the second sidewall segment (along the widest plane) is approximately 2 mm to 25 mm. In one embodiment, the diameter of the second sidewall segment (along the widest plane) is approximately 2 mm to 20 mm. In one embodiment, the diameter of the second sidewall segment (along the widest plane) is approximately 2 mm to 15 mm. In one embodiment, the diameter of the second sidewall segment (along the widest plane) is approximately 2 mm to 10 mm. In one embodiment, the diameter of the second sidewall segment is approximately 3 mm to 15 mm.

[0020] The cell culture device, particularly its well, may further include a third side wall segment connected to the second side wall segment by a second ledge. The third side wall segment defines the boundary of the space above the second volume, such that the planes of the third side wall segment and the second ledge define the boundary of a third volume that can hold or store liquid and / or a polymerizable liquid (under the condition that the first and second volumes are filled with liquid or a polymerizable liquid).

[0021] In one embodiment, the third side wall portion extends from the second ledge towards the opening. In one embodiment, the third side wall portion extends from the second ledge to the opening.

[0022] In one embodiment, the height of the third side wall segment is greater than the height of the second side wall segment, and the third volume is greater than the second volume. In one embodiment, the height of the third side wall segment is about 1 - 20 mm (±1 mm), about 1 - 15 mm (±1 mm), or about 1 - 10 mm (±1 mm).

[0023] In one embodiment, the second ledge and the third side wall segment form an obtuse angle. In one embodiment, the angle is 90 degrees to 100 degrees.

[0024] In one embodiment, the width of the second ledge is about 0.2 mm to about 5 mm.

[0025] In one embodiment, the third side wall segment is circular or substantially circular in the plane of the bottom wall (e.g., when looking from the opening towards the bottom wall). In one embodiment, the diameter of the third side wall segment at any position along its height is greater than the diameter of the second side wall segment at any position along its height.

[0026] The cell culture device of the present disclosure may further include a plurality of wells. In one embodiment, each well is substantially as described above.

[0027] Thus, in another aspect of the present disclosure, a cell culture device is provided that includes a plurality of wells having a bottom wall and an opening. In certain embodiments, two or more of the plurality of wells, or all of the plurality of wells, may include a first sidewall segment that is connected to the bottom wall and extends away from the bottom wall. The first sidewall segment and the bottom wall define a boundary of a first volume for holding a liquid and / or a polymerizable liquid.

[0028] The cell culture device of this aspect may further include a second sidewall segment connected to the first sidewall segment by a first ledge. The second sidewall segment defines a boundary of a space above the first volume, such that the plane of the second sidewall segment and the first ledge defines a boundary of a second volume that can hold or store a liquid and / or a polymerizable liquid (under the condition that the first volume is filled with a liquid or a polymerizable liquid).

[0029] In one embodiment, the height of the first sidewall segment is greater than the height of the second sidewall segment.

[0030] In one embodiment, the first volume is equal to or substantially equal to the second volume.

[0031] In another aspect of the present disclosure, a method of culturing cells is provided that includes seeding a cell population in a well of the cell culture device described herein.

[0032] The method of the present disclosure may further include adding a liquid extracellular matrix, one or more liquid extracellular matrix proteins, or a liquid hydrogel to a first volume surrounded by the first sidewall segment of the cell culture device. In one embodiment, adding a liquid extracellular matrix, one or more liquid extracellular matrix proteins, or a liquid hydrogel includes filling the first volume surrounded by the first sidewall segment with a liquid extracellular matrix, one or more liquid extracellular matrix proteins, or a liquid hydrogel.

[0033] The methods of the present disclosure may further include polymerizing a liquid extracellular matrix, one or more liquid extracellular matrix proteins, or a liquid hydrogel, or enabling the polymerization of a liquid extracellular matrix, one or more liquid extracellular matrix proteins, or a liquid hydrogel.

[0034] In one embodiment, the cell population is suspended and / or embedded in an extracellular matrix, one or more extracellular matrix proteins, or a hydrogel.

[0035] The method of the present disclosure may further include exposing or bringing a cell population to a cell culture medium by adding the cell culture medium to a second volume enclosed by a second sidewall segment. In one embodiment, the second volume enclosed by the second sidewall segment is filled with cell culture medium.

[0036] In one embodiment, the volume of the extracellular matrix, one or more extracellular matrix proteins, or hydrogel is equal to or substantially equal to the volume of the cell culture medium.

[0037] The methods of this disclosure may further include forming or generating multicellular aggregates or organoids from a population of cells.

[0038] The methods of this disclosure may further include assaying cell populations. In one embodiment, the assay may be an automated assay, for example, via an automated liquid handler and / or an automated imager. The types of assays performed using the above apparatus or by carrying out the above methods are not particularly limited and include cytotoxicity assays, compound screening assays, growth condition optimization assays, differentiation assays, and the like.

[0039] To better understand the various embodiments described herein and to more clearly illustrate how these various embodiments can be carried out, the accompanying drawings showing at least one exemplary embodiment are referred to below as examples. The drawings are not intended to limit in any way the scope of the teachings described herein. [Brief explanation of the drawing]

[0040] [Figure 1] Embodiments of the cell culture apparatus of this disclosure are shown in an oblique cross-sectional view (A) and a top view (B) showing a cell culture in a first volume. The darker shadow in (A) may correspond to the extracellular matrix or extracellular matrix components, and the gradually fading shadow above it may correspond to the cell culture medium or a second layer of extracellular matrix or extracellular matrix components. [Figure 2] This is an enlarged view showing an embodiment of the wells included in the cell culture apparatus of the present disclosure, in a top view (A) and a separated cross-sectional view (B) along line AA. [Figure 3] This is an enlarged view showing different embodiments of the wells included in the cell culture apparatus of the present disclosure, in a top view (A) and a separated cross-sectional view (B) along line AA. [Figure 4] Further different embodiments of the wells included in the cell culture apparatus of this disclosure are shown in a top view (A) and an enlarged view of a separated cross-section along line AA (B). [Figure 5] This is a line graph showing the growth of multicellular structures in different embodiments of the cell culture apparatus of this disclosure. The growth of human organoids of liver (A) and colon (B) in the extracellular matrix was evaluated over several days. The height (depth) of the first sidewall portion changed as shown. The growth of organoids in the cell culture apparatus of this disclosure is shown in comparison to the growth of organoids in standard dome culture. Data are expressed as mean ± SD. [Figure 6]The characteristics of multicellular structures grown in the cell culture apparatus of this disclosure are shown. Line graph (A) shows the growth of colon organoids over time in either the cell culture apparatus of this disclosure ("Improved") or a standard dome culture ("Dome Type"). Data are mean ± SD, n=4, p<0.001, based on two-way Tukey analysis of variance for multiple comparisons. Graph showing the plating efficiency of colon organoids seeded in either the cell culture apparatus of this disclosure or a standard dome culture after 7 days of culture. Plating efficiency was calculated by dividing the number of clumps seeded in D0 by the number of organoids with a diameter greater than 100 μm, counted after 7 days of culture (B). Data are mean ± SD, n=4, p<0.001, based on a t-test. Histogram (C) shows the size distribution of human intestinal organoids after 7 days of culture in either the cell culture apparatus of this disclosure or a standard dome culture. [Figure 7] This is a line graph showing the responsiveness of organoids to forskolin. The graphs show the expansion rates of normal human intestinal organoids in either the 24-well cell culture apparatus of this disclosure or a standard dome culture, after approximately 90 minutes of exposure to 5 μM forskolin (FSK) (A) or DMSO control (B). Data represent mean ± SD, n=4. The graphs also show the expansion rates of normal human intestinal organoids in either the 96-well cell culture apparatus of this disclosure or a standard dome culture, after approximately 90 minutes of exposure to different doses of forskolin (FSK) (C). Data represent mean ± SD, n=3, p<0.01, based on two-way ANOVA. [Figure 8] Representative images of human liver organoids formed / differentiated in various concentrations of extracellular matrix are shown. Organoids were seeded on 100%, 75%, 50%, 20%, or 10% Matrigel mixed with culture medium and added to the 24-well plate cell culture apparatus of this disclosure or as dome cultures on standard 24-well plates, and imaged on day 4 (A) and day 7 (B). Scale bars are 1000 μm in (A) and 200 μm in (B). [Figure 9]The graphs shown quantify the evaporation levels from the wells of the 24-well (A) or 96-well (B) cell culture apparatus of this disclosure compared to the corresponding wells of standard 24-well and 96-well plates. Each data point represents a replication of the experiment, and the horizontal dashed line represents the mean ± SD. [Modes for carrying out the invention]

[0041] This disclosure relates to cell culture, and more specifically to apparatus for cell culture, and uses or methods of using such apparatus for cell culture. In certain embodiments, apparatus for culturing cells is described. In certain embodiments, methods or processes of cell culture using the apparatus of this disclosure are described.

[0042] Various apparatuses, systems, and methods are described below to provide examples of at least one embodiment of the claimed subject matter. The embodiments described below are not limiting to the claimed subject matter, and any claimed subject matter may cover apparatuses, systems, and methods different from those described below. The claimed subject matter is not limited to systems, apparatuses, and methods having all the features of any of the systems, apparatuses, or methods described below, nor is it limited to features common to some or all of the systems, apparatuses, and methods described below. Possible claimed subject matter may exist in any combination or subcombination of elements or process steps disclosed in any part of this document, including the claims and figures. A person skilled in the art will therefore understand that a system, apparatus, or method disclosed pursuant to the teachings herein may embody one or more of the features contained herein, and that such features can be used in any particular combination or subcombination that is physically feasible and feasible for its intended purpose.

[0043] Furthermore, the systems, apparatus, or methods described below may not be embodiments of the claimed subject matter. Subject matter disclosed in the systems, apparatus, or methods described herein and not claimed in this document may be subject to other means of protection, such as ongoing patent applications, and the applicant(s), inventor(s), and / or owner(s) have no intention of abandoning, disclaiming, or providing to the public such inventions by disclosure in this document.

[0044] Furthermore, it will be understood that, in order to make the explanation concise and clear, reference numbers may be repeated between figures to indicate corresponding or similar elements, where deemed appropriate. In addition, many specific details are given in order to fully understand the exemplary embodiments described herein. However, those skilled in the art will understand that the exemplary embodiments described herein can be carried out without these specific details. In other examples, details of well-known methods, procedures, and components are not described in order to avoid obscuring the exemplary embodiments described herein. Furthermore, this description should not be construed as limiting the scope of the exemplary embodiments described herein.

[0045] It should be noted that terms of degree used herein, such as “substantially,” “about,” and “approximately,” mean a reasonable deviation of the modified term that does not substantially alter the result. These terms of degree should be interpreted as including deviations of the modified term such as 1%, 2%, 5%, 10%, or ±0.1mm, ±0.5mm, ±1mm, unless they negate the meaning of the modified term.

[0046] Furthermore, descriptions of numerical ranges by endpoints in this specification include all numbers and fractions within that range (for example, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). Also, all numbers and their fractions are considered to be modified by the term “approximately.” This term means a change to a numerical value of a specific quantity referenced, such as 1%, 2%, 5%, 10%, or ±0.1 mm, ±0.5 mm, ±1 mm, etc., where the final result does not change significantly.

[0047] Furthermore, it should be noted that the phrase "and / or" as used herein is intended to represent an all-encompassing "or." That is, "X and / or Y" is intended to mean, for example, X or Y, or both. As a further example, "X, Y, and / or Z" is intended to mean X or Y or Z, or any combination thereof.

[0048] As used in this disclosure, the terms “cell culture” or “culturing cells” refer to the in vitro or ex vivo proliferation, growth, maintenance, self-organization, differentiation, processing, and / or assay of one or more cells in a container or vessel. Cell culture typically involves or is carried out in a culture environment, which includes a culture medium. The cell culture environment may further include an extracellular matrix, one or more extracellular matrix proteins, a (synthetic) hydrogel, or a layer of feeder cells.

[0049] As used in this disclosure, the terms “extracellular matrix,” “extracellular matrix protein,” or “ECM” refer to molecules that provide structural and / or biochemical support to surrounding cells. The extracellular matrix may include interlocking meshes of fibrous proteins and / or polysaccharide glycosaminoglycans. ECMs are also widely known to promote cell adhesion, intercellular communication, and differentiation within certain tissues or cell aggregates. In this disclosure, both innate (e.g., produced or secreted by cells) and synthetic (e.g., synthesized from chemical and / or protein components) extracellular matrix are considered. The extracellular matrix may include one or more extracellular matrix proteins. The components of the extracellular matrix, and by extension the extracellular matrix within the scope of this application, may include one or more of the following: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosine, anchorin, chondronectin, link proteins, bone sialoproteins, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, kalinin, synthetic polymer-based hydrogels (e.g., polyethylene glycol, polyvinyl, or any derivative or analog thereof), plant-based hydrogels (e.g., cellulose, hemicellulose, lignin, starch, pectin, or any derivative or analog thereof), proteoglycans (e.g., heparan sulfate, chondroitin sulfate, keratin sulfate), glycosaminoglycans, or hyaluronic acid. In one embodiment, the extracellular matrix or matrix component described herein (which may be used in or with the culture medium) may be a mixture of gelatinous proteins and / or polysaccharides secreted by cells such as fibroblasts, chondrocytes, or Engelblesse-Holm swarm (EHS) mouse sarcoma cells. In some embodiments, the extracellular matrix is ​​Matrigel. The ECM proteins of this disclosure may be coated onto cell culture surfaces such as the bottom walls of containers, dishes, flasks, or plates.

[0050] As used in this disclosure, the term “organoid” refers to a multicellular structure that may be generated in vitro or ex vivo from an initiating cell population, such as primary cells or pluripotent stem cells. Organoids tend to exhibit a higher level of tissue, reminiscent of the tissue observed in the corresponding tissue. Organoids corresponding to various tissue types can be formed using, for example, kits and protocols commercialized by STEMCELL Technologies. An exemplary organoid kit is available from IntestiCult. TM HepatiCult TM This includes kits for STEMdiff® Intestinal Organoid or STEMdiff® Bloodvessel Organoid. While this disclosure primarily focuses on epithelial organoids, it is not necessarily limited to epithelial organoids. Examples of epithelial tissues that can be formed into organoids of this disclosure (according to the media and methods disclosed herein) include colon organoids, liver organoids, intestinal organoids, pancreatic organoids, and the like.

[0051] Device One aspect of this disclosure provides a cell culture apparatus, in other words, an apparatus for culturing cells.

[0052] Referring to Figures 1 to 4, the cell culture apparatus 1 of this disclosure includes a well 5 having an opening 7 and a bottom wall 9. The well 5 may further include one or more side walls 11 connecting the bottom wall 9 to the (opposite) opening 7. In some embodiments, the cell culture apparatus 1 includes a plurality of such wells 5.

[0053] The cell culture apparatus 1 is not limited in terms of its constituent or forming materials, as long as they are not toxic to the cell culture. In one embodiment, the cell culture apparatus 1 is made of a polymer or plastic such as polystyrene, polymethylpentene, acrylic, polypropylene, or polycarbonate. In one embodiment, the cell culture apparatus 1 is made of glass or silicate. In one embodiment, different parts of the cell culture apparatus 1 are made of different materials. For example, the base of the cell culture apparatus may be made of glass or silicate, and the well definition members attached to the base may be made of polymer or plastic, or vice versa. In a preferred embodiment, the cell culture apparatus is made of a single, integrated structure suitable for injection molding or 3D printing, such as plastic or polymer.

[0054] The side wall 11 of the cell culture apparatus 1 may be organized into a plurality of side wall segments or portions 13, each side wall segment or portion cooperating to connect the bottom wall 9 to the opening 7. The plurality of side wall segments 13 may include a first side wall segment 15 and a second side wall segment 25. Each of the first side wall portion 15 and the second side wall portion 25 may be continuous, that is, the first side wall portion 15 and the second side wall portion 25 may be made of a single material and form the perimeter of the well 5.

[0055] The cross-sectional shape of the first side wall segment 15 (cut in the plane of the bottom wall 9 and / or opening 7) is not particularly limited. In one embodiment, the cross-sectional shape of the first side wall segment 15 is rounded or circular when viewed along the axis from the opening 7 to the bottom wall 9 (for example, in the plane of the bottom wall 9 and / or opening 7).

[0056] The cross-sectional shape of the second side wall segment 25 (cut in the plane of the bottom wall 9 and / or opening 7) is not particularly limited. In certain embodiments, the cross-sectional shape of the first side wall segment 15 is the same as the cross-sectional shape of the second side wall segment 25. In one embodiment, the cross-sectional shape of the second side wall portion 25 is rounded or circular when viewed along the axis from the opening 7 to the bottom wall 9 (for example, in the plane of the bottom wall 9 and / or opening 7).

[0057] In certain embodiments, if the first side wall segment 15 and the second side wall segment 25 are rounded or circular, they are concentric.

[0058] In the case of a circular shape, the diameter of the first side wall segment 15 (at the widest plane or distance between opposing walls) may be approximately 2 mm to 35 mm, approximately 2.25 mm to 30 mm, approximately 2.5 mm to 25 mm, approximately 2.75 mm to 20 mm, or approximately 3 mm to 15 mm. In one embodiment, the diameter of the first side wall segment 15 (at the widest plane or distance between opposing walls) is approximately 2.5 mm to 10 mm (±1 mm), or approximately 3 mm to 9 mm (±1 mm).

[0059] In the case of a circular shape, the diameter of the second side wall segment 25 (at the widest plane or distance between opposing walls) may be approximately 1 mm to 40 mm, approximately 1.25 mm to 35 mm, approximately 1.5 mm to 30 mm, approximately 1.75 mm to 25 mm, or approximately 2 mm to 20 mm. In one embodiment, the diameter of the second side wall segment 25 (at the widest plane or distance between opposing walls) is approximately 4 mm to 13 mm (±1 mm), or approximately 5 mm to 12 mm (±1 mm). In one embodiment, the diameter of the second side wall segment 25 (at the widest plane or distance between opposing walls) is approximately 5 mm to 33 mm, or approximately 3.5 mm to 13 mm, or approximately 4 mm to 12 mm.

[0060] In embodiments where well 5 is configured in a 96-well plate format, the diameter of the first sidewall segment 15 (at its widest plane or between opposing walls) may be approximately 2 mm to approximately 5 mm (±0.5 mm), approximately 2.5 mm to approximately 4.5 mm (±0.5 mm), or approximately 3 mm to approximately 4 mm (±0.5 mm). In such embodiments, the diameter of the second sidewall segment 25 may be approximately 3 mm to approximately 7 mm (±0.5 mm), approximately 3.5 mm to approximately 6 mm (±0.5 mm), or approximately 4 mm to approximately 5 mm (±0.5 mm).

[0061] In embodiments where well 5 is configured in a 24-well plate format, the diameter of the first sidewall segment 15 (at its widest plane or between opposing walls) may be approximately 5 mm to approximately 12 mm (±0.5 mm), approximately 5.5 mm to approximately 11 mm (±0.5 mm), approximately 6 mm to approximately 10 mm (±0.5 mm), or approximately 5.5 mm to approximately 9.5 mm (±0.5 mm). In such embodiments, the diameter of the second sidewall segment 25 may be approximately 7.5 mm to approximately 15 mm (±0.5 mm), approximately 8.5 mm to approximately 14 mm (±0.5 mm), approximately 9 mm to approximately 13.5 mm (±0.5 mm), approximately 9.5 mm to approximately 13 mm (±0.5 mm), or approximately 10 mm to approximately 12.5 mm (±0.5 mm).

[0062] In some embodiments, the well 5 is comprised of a plate form other than 96-well or 24-well, such as a 48-well plate, a 12-well plate, a 6-well plate, or a Petri dish, in which case the diameters of the first sidewall portion 15 and the second sidewall portion 25 may be as described above and may be adjusted accordingly.

[0063] The first sidewall segment 15 is connected to the bottom wall 9 and extends therefrom in a direction away from the bottom wall 9, for example toward or toward the opening 7. Thus, the first sidewall segment 15 encloses the bottom wall 7, and together the first sidewall segment 15 and the bottom wall 9 define the boundary of a first volume v1 for storing / holding liquids and / or polymerizable liquids, such as liquids containing extracellular matrix or extracellular matrix proteins.

[0064] The height h1 of the first sidewall segment 15 is limited only by the dimensions of the cell culture apparatus 1, more specifically by the depth of the well 5. In one embodiment, the height h1 of the first sidewall segment 15 is approximately 0.5 mm to 5 mm, approximately 0.6 mm to 4 mm, approximately 0.7 mm to 3 mm, approximately 0.8 mm to 2.5 mm, or approximately 0.9 mm to 2 mm.

[0065] In some embodiments, the height h1 of the first sidewall segment 15 is the same or substantially the same regardless of whether the well 5 is composed of a 96-well plate or a 24-well plate. In some embodiments, the height h1 of the first sidewall segment 15 differs depending on whether the well 5 is composed of a 96-well plate or a 24-well plate. In one embodiment, the height h1 of the first sidewall segment 15 is greater when the well 5 is composed of a 24-well plate compared to when the well 5 is composed of a 96-well plate.

[0066] In some embodiments, the well 5 is comprised of a plate form other than 96-well or 24-well, such as a 48-well plate, a 12-well plate, a 6-well plate, or a Petri dish, in which case the height h1 of the first side wall segment 15 and the second side wall segment 25 may be as described above, adjusted accordingly, or different.

[0067] When the cell culture apparatus 1 is viewed perpendicular to the depth axis d of the well 5, the second sidewall segment 25 is located above the first sidewall segment 15. However, the second sidewall segment 25 is connected to the first sidewall segment 15 by a first ledge 30 that extends outward from the center point of the bottom wall 9. At the point where the second sidewall segment connects to the first ledge 30, the second sidewall segment 25 extends toward or in the direction toward the opening 7.

[0068] Therefore, in certain embodiments, the second side wall segment 25 is dimensionally wider than the first side wall segment 15 (when measured in the plane of the bottom wall 9 and / or opening 7). Regardless of the shape of the first and second side wall segments (as viewed from the plane of the bottom wall 9 and / or opening 7), unless otherwise specified, for the purposes of this explanation, the distance between two points on each side wall segment is measured at the point where the measurement is maximum. Thus, in the case of a rectangle, it is measured along the longest side, and in the case of a circle, it is measured along the diameter. Thus, in the case of a circle, the diameter of the second side wall segment 25 is greater than the diameter of the first side wall segment 15.

[0069] In one embodiment, the cross-sectional shape of the second side wall segment is a polygon such as a quadrilateral, pentagon, hexagon, or octagon (viewed along the axis from the opening 7 to the bottom wall 9). In another embodiment, the cross-sectional shape of the second side wall segment is rounded or circular (viewed along the axis from the opening 7 to the bottom wall 9).

[0070] If the apparatus 1 includes a first sidewall segment 15 and a second sidewall segment 25, the first ledge 30 connects the bottom edge 32 (i.e., the edge closest to the bottom wall 9) of the second sidewall segment 25 to the top edge 34 (i.e., the edge furthest from the bottom wall 9) of the first sidewall segment. In one embodiment, the width w1 of the first ledge may be about 0.1 mm to about 5 mm (±0.5 mm), about 0.2 mm to about 4 mm (±0.5 mm), about 0.3 mm to about 3 mm (±0.5 mm), or about 0.4 mm to about 2 mm (±0.5 mm).

[0071] In embodiments where well 5 is configured in a 96-well plate format, the width of the first ledge w1 may be approximately 0.1 mm to approximately 1 mm (±0.5 mm), approximately 0.2 mm to approximately 0.9 mm (±0.5 mm), approximately 0.3 mm to approximately 0.8 mm (±0.5 mm), or approximately 0.4 mm to approximately 0.7 mm (±0.5 mm).

[0072] In embodiments where well 5 is configured in a 24-well plate format, the width of the first ledge w1 may be approximately 0.5 mm to approximately 5 mm (±0.5 mm), approximately 0.6 mm to approximately 4 mm (±0.5 mm), approximately 0.7 mm to approximately 3 mm (±0.5 mm), or approximately 0.8 mm to approximately 2 mm (±0.5 mm).

[0073] In some embodiments, the well 5 is comprised of a plate format other than a 96-well or 24-well plate, such as a 48-well plate, a 12-well plate, a 6-well plate, or a Petri dish, and the width w1 of the first ledge may be as described above or adjusted accordingly.

[0074] The height h2 of the second sidewall segment 25 is limited only by the dimensions of the cell culture apparatus 1, more specifically by the depth of the well 5. In some embodiments, the height h2 of the second sidewall segment depends on the height h1 of the first sidewall segment 15. In one embodiment, the height h2 of the second sidewall segment 25 is about 0.2 mm to about 2 mm, about 0.3 mm to about 1.5 mm, or about 0.4 mm to about 1 mm. In one embodiment, the height h1 of the first sidewall segment 15 is greater than the height h2 of the second sidewall segment 25.

[0075] In fact, the second sidewall segment 25 defines the boundary of a second volume v2 for holding a liquid and / or polymerizable liquid. More specifically, the planes of the second sidewall segment 25 and the first ledge 30 define the boundary of the second volume v2; in other words, the space above the first volume v1 enclosed by the second sidewall segment 25 constitutes the second volume v2. In one embodiment, the first volume v1 is equal to the second volume v2. In one embodiment, the first volume v1 has an error of ±10% or less compared to the second volume v2.

[0076] The first side wall segment 15 may extend upward perpendicularly from the bottom wall 9 toward or in the direction of the opening 7. Preferably, the first side wall segment 15 is obtuse with respect to the bottom wall 9 (e.g., 91°, 92°, 93°, 94°, 95°, 96°, 97°, 98°, 99° or more, or 99°, 97°, 95°, or less than 93°). The second side wall segment 25 may extend upward perpendicularly from the first ledge 30 (or with respect to the plane of the bottom wall 9) toward or in the direction of the opening 7. Preferably, the second side wall segment 25 is obtuse with respect to the first ledge 30 (or with respect to the plane defined by the bottom wall 9) (e.g., 91°, 92°, 93°, 94°, 95°, 96°, 97°, 98°, 99° or more, or 99°, 97°, 95°, or less than 93°). In one embodiment, the first sidewall segment 15 forms an obtuse angle with the bottom wall 9, and the second sidewall segment 25 forms an obtuse angle with the first ledge 30 (or with the plane defined by the bottom wall 9). In one embodiment, only one of the first sidewall segment 15 and the second sidewall segment 25 forms an obtuse angle with the bottom wall 9 or the first ledge 30, respectively, while the other extends perpendicular to the plane of the bottom wall 9.

[0077] The cell culture apparatus 1 may further include a third sidewall segment 35. In one embodiment, when the cell culture apparatus 1 is viewed perpendicular to the depth axis d of the well 5, the third sidewall segment 35 is located above the first sidewall segment 15 and immediately above the second sidewall segment 25. Nevertheless, the third sidewall segment 35 is connected to the second sidewall segment 25 by a second ledge 40 that extends outward from the second sidewall segment 25 with respect to the center point of the bottom wall 9. At the point where the third sidewall segment 35 connects to the second ledge 40, the third sidewall segment 35 extends toward or in the direction toward the opening 7.

[0078] Therefore, the third side wall segment 35 is dimensionally wider than the first side wall segment 15 and the second side wall segment 25 (when measured in the plane of the bottom wall 9 and / or opening 7). Regardless of the cross-sectional shape of the first, second, or third side wall segment (the cross-sectional shape viewed from the direction of the opening 7 toward the bottom wall 9), unless otherwise specified, the distance between two points on each side wall segment is measured at the point where the measurement is maximum. Therefore, in the case of a rectangle, it is measured along the longest side, and in the case of a circle, it is measured along the diameter. Therefore, if the cross-sectional shape of the third side wall segment is circular, the diameter of the third side wall segment 35 may be larger than the diameter of the second side wall segment 25 (and the first side wall segment 15).

[0079] In one embodiment, the diameter of the third side wall segment 35 is approximately 5 mm to 40 mm (±1 mm), approximately 5.25 mm to 35 mm (±1 mm), approximately 5.5 mm to 30 mm (±1 mm), approximately 5.75 mm to 30 mm (±1 mm), approximately 6 mm to 30 mm (±1 mm), or approximately 6 mm to 16 mm (±1 mm).

[0080] In an embodiment in which well 5 is configured in a 96-well plate format, the diameter of the third sidewall segment 35 may be approximately 3 mm to approximately 9 mm (±1 mm), approximately 4 mm to approximately 8 mm (±1 mm), or approximately 5 mm to approximately 7 mm (±1 mm).

[0081] In an embodiment in which well 5 is configured in a 24-well plate format, the diameter of the third sidewall segment 35 may be approximately 10 mm to approximately 20 mm (±1 mm), approximately 11 mm to approximately 19 mm (±1 mm), approximately 12 mm to approximately 18 mm (±1 mm), or approximately 13 mm to approximately 17 mm (±1 mm).

[0082] In some embodiments, the well 5 is comprised of a plate form other than a 96-well or 24-well plate, such as a 48-well plate, a 12-well plate, a 6-well plate, or a Petri dish, in which case the diameter of the third side wall segment 35 may be as described above and may be adjusted accordingly.

[0083] In one embodiment, the third side wall segment is a polygon such as a quadrilateral, pentagon, hexagon, or octagon (viewed along the axis from the opening 7 to the bottom wall 9). In another embodiment, the third side wall segment is rounded or circular (viewed along the axis from the opening 7 to the bottom wall 9).

[0084] The second ledge 40 connects the bottom edge 42 (i.e., the edge closest to the bottom wall 9) of the third side wall segment 35 to the top edge 44 (i.e., the edge furthest from the bottom wall 9) of the second side wall segment 25. In one embodiment, the width w2 of the second ledge may be about 0.2 mm to about 5 mm, about 0.4 mm to about 4 mm, about 0.6 mm to about 3 mm, or about 0.7 mm to about 2.5 mm.

[0085] In embodiments where well 5 is configured in a 96-well plate format, the width w2 of the second ledge may be approximately 0.2 mm to approximately 1.5 mm, approximately 0.4 mm to approximately 1.25 mm, or approximately 0.6 mm to approximately 1 mm.

[0086] In embodiments in which well 5 is configured in a 24-well plate format, the width w1 of the second ledge may be approximately 0.5 mm to approximately 5 mm, approximately 0.75 mm to approximately 4 mm, approximately 1 mm to approximately 3 mm, or approximately 1.25 mm to approximately 2 mm.

[0087] In some embodiments, the well 5 is comprised of a plate format other than a 96-well or 24-well plate, such as a 48-well plate, a 12-well plate, a 6-well plate, or a Petri dish, and the width w2 of the second ledge may be as described above or adjusted accordingly.

[0088] The height h3 of the third sidewall segment 35, if present, is limited only by the dimensions of the cell culture apparatus 1, more specifically by the depth of the well 5. In one embodiment, the height h3 of the third sidewall 35 depends on the sum of the height h1 of the first sidewall segment 15 and the height h2 of the second sidewall segment 25. In one embodiment, the height h3 of the third sidewall segment 35 is about 1 mm to about 16 mm, about 1.5 mm to about 9 mm, about 2 mm to about 8.5 mm, or about 2.25 mm to about 7 mm. In one embodiment, the height h3 of the third sidewall segment 35 is greater than the height h2 of the second sidewall segment 25. In one embodiment, the height h3 of the third sidewall segment 35 is greater than the height h1 of the first sidewall segment 15. In one embodiment, the height h3 of the third sidewall segment 35 is greater than the sum of the height h2 of the second sidewall segment 25 and the height h1 of the first sidewall segment 15.

[0089] Similar to the first and second sidewall segments 15 and 25, the third sidewall segment 35 defines the boundary of a third volume v3 for holding a liquid and / or polymerizable liquid. More specifically, the planes of the third sidewall segment 35 and the second ledge 40 define the boundary of the third volume v3, in other words, the space above the second volume v2 enclosed by the third sidewall segment 35 constitutes the third volume v3. In one embodiment, the third volume v3 is larger than the second volume v2. In one embodiment, the third volume v3 is larger than the first volume v1. In one embodiment, the third volume v3 is larger than the sum of the second volume v2 and the first volume v1.

[0090] The third side wall segment 35 extends toward or in the direction of the opening 7 perpendicular to the plane of the bottom wall 9 and / or the second ledge 40. Preferably, the third side wall segment 35 forms an obtuse angle with respect to the plane of the bottom wall 9 and / or the second ledge 40. In one embodiment, each of the first side wall segment 15, the second side wall segment 25, and the third side wall segment 35 forms an obtuse angle with respect to the plane of the bottom wall 9. In one embodiment, only one or two of the first side wall segment 15, the second side wall segment 25, and the third side wall segment 35 form an obtuse angle with respect to the plane of the bottom wall 9, while the other side wall segments extend perpendicular to the plane of the bottom wall 9.

[0091] The above description focuses on the features and structure of a single well 5 of the cell culture apparatus 1, but it is within the scope of this disclosure that the cell culture apparatus 1 may include multiple such wells. In one embodiment, the cell culture apparatus 1 is sized substantially in accordance with American National Standards Institute (ANSI) standards. In one embodiment, the cell culture apparatus 1 is a 96-well plate, each of which includes some of the features and characteristics of the embodiments described above. In one embodiment, the cell culture apparatus 1 is a 24-well plate, each of which includes some of the features and characteristics of the embodiments described above. In one embodiment, the cell culture apparatus 1 is a 6-well plate, each of which includes some of the features and characteristics of the embodiments described above.

[0092] The cell culture apparatus 1 may further include a lid. In one embodiment, the lid and apparatus 1 are designed to reduce or limit evaporation from the wells 5, particularly from the edge wells and corner wells. Evaporation causes changes in the osmotic pressure of the culture medium contained in the wells 5, or harmful changes in the concentration of nutrients and waste products. In one embodiment, evaporation is reduced or limited by improving the fit between the lid and the lid support surface of the cell culture apparatus 1, for example, by minimizing the space between them.

[0093] As will be further described below, the cell culture apparatus 1 can be used for a variety of cell culture applications. In one embodiment, cells may be seeded directly onto the bottom wall 9, and then cell culture medium may be added to the well 5, for example, up to the level of the first ledge 30 or the second ledge 40, or (in embodiments including a third wall 35) beyond that level. In one embodiment, at least the bottom wall 9 is tissue culture treated. In another embodiment, at least the bottom wall 9 is not tissue culture treated. In such embodiments, depending on the type of cells cultured at the bottom, the wall 9 may be coated with an extracellular matrix, at least one extracellular matrix protein, or a hydrogel.

[0094] In one embodiment, extracellular matrix (or at least one extracellular matrix protein or hydrogel) may be added to a first volume v1 and cells may be seeded thereon. In one embodiment, extracellular matrix (or at least one extracellular matrix protein) and one or more cells may be added to the first volume v1 and cells may be seeded thereon. In one embodiment, the extracellular matrix (or at least one extracellular matrix protein) is filled up to a first ledge 30 or a second ledge 40.

[0095] Because each extracellular matrix has a different gelation mechanism, specific challenges arise depending on the type of extracellular matrix and the type of cell culture vessel or container used. In fact, certain extracellular matrix products polymerize in a temperature-dependent manner, such as Matrigel. TM It is a liquid at low temperatures and polymerizes at high temperatures. Therefore, depending on the rate of temperature increase, Matrigel will be distributed on or inside a container or vessel. TM The shape and localization of thermally gelling substrates such as these are unpredictable. Furthermore, in the case of extracellular substrates that polymerize by photocrosslinking, chemical activation, enzymatic action, or introduction of complementary interaction domains, the flat culture surface to which they are applied does not maintain the desired shape until polymerization is induced.

[0096] An advantage of the cell culture apparatus 1 of this disclosure is that the first sidewall segment 15 and / or the second sidewall segment 25 restrict the shape of the polymerized extracellular matrix protein. Indeed, the extracellular matrix is ​​surrounded by the sidewall segments, but the height of the extracellular matrix at any point along the surface region exposed to air (before culture medium is added) can also be controlled when a given volume of extracellular matrix fills the first volume v1 (or the first volume v1 and the second volume v2).

[0097] Another advantage of the cell culture apparatus of this disclosure is that variability due to mass transfer rates is limited or eliminated by controlling the shape and height of the polymerized extracellular matrix. As a result, three-dimensional cell assemblies or constructs, such as organoids, formed / induced therein can be more uniform in size compared to the current state of dome approaches. Furthermore, three-dimensional cell assemblies or constructs, such as organoids, formed / induced in the cell culture apparatus of this disclosure may also exhibit improved seeding efficiency, proliferation, and / or function.

[0098] Another advantage of the cell culture apparatus of this disclosure is that the predictability of cell culture location and the reduction or elimination of meniscus effects, along with the restricted localization of the region and volume enclosed by the first sidewall segment 15, enable reliable imaging.

[0099] method Other embodiments of this disclosure provide a method or process for culturing or assaying cells using the cell culture apparatus 1 described above. In certain embodiments, the method or process relates to culturing or assaying cells using the cell culture apparatus 1 under non-adherent conditions, in three dimensions, or embedded in polymerized extracellular matrix, extracellular matrix proteins, or hydrogels. In one embodiment, the cells in culture are multicellular aggregates or organoids, and such aggregates / organoids may grow or form during culture within the cell culture apparatus 1.

[0100] In one embodiment, the method of the present disclosure includes seeding a cell population into well 5 of a cell culture apparatus 1.

[0101] The cell populations that can be cultured according to the methods / processes of this disclosure are not particularly limited. In certain embodiments, cells require or benefit from exposure to extracellular matrix, extracellular matrix components, or hydrogels. In one embodiment, the cells cultured according to the methods / processes of this disclosure are mammalian cells, such as human, non-human primate, or rodent cells.

[0102] In one embodiment, the cell population cultured according to the method / process of the Disclosure is mammalian epithelial cells, e.g., mammalian epithelial stem cells or progenitor cells. In one embodiment, mammalian epithelial stem cells or progenitor cells form organoids when cultured in the cell culture apparatus of the Disclosure according to the method disclosed herein. In one embodiment, mammalian epithelial stem cells or progenitor cells form organoids while embedded in extracellular matrix, extracellular matrix components, or hydrogel added to the cell culture apparatus of the Disclosure.

[0103] In one embodiment, the cell population cultured according to the method / process of the disclosure is primary, i.e., obtained from a patient, subject, biopsy, or otherwise. In one embodiment, the cells cultured according to the method / process of the disclosure are pluripotent stem cells (PSCs), e.g., induced PSCs or embryonic stem cells. In one embodiment, the cells cultured according to the method / process of the disclosure are PSC-derived, i.e., the cells were undifferentiated pluripotent stem cells at some point in time, but subsequently differentiated into the downstream lineage of interest (e.g., epithelial-like stem cells, in a non-limiting example).

[0104] The method of the present disclosure may further include adding a liquid extracellular matrix, one or more liquid extracellular matrix proteins, or a liquid hydrogel to a first volume v1 enclosed by a first sidewall segment 15. In certain embodiments, the first volume v1 is filled with a liquid extracellular matrix, one or more liquid extracellular matrix proteins, or a liquid hydrogel. In such embodiments, the liquid extracellular matrix, one or more liquid extracellular matrix proteins, or liquid hydrogel may be added up to the level of a first ledge 30.

[0105] In one embodiment, a liquid extracellular matrix, one or more liquid extracellular matrix proteins, or a liquid hydrogel may be added to or filled beyond a first volume v1, for example, into a second volume v2. In one embodiment, the second volume v2 is filled with a liquid extracellular matrix, one or more liquid extracellular matrix proteins, or a liquid hydrogel. In one embodiment, the first volume v1 and the second volume v2 are sequentially filled with the same or different liquid extracellular matrix, one or more liquid extracellular matrix proteins, or a liquid hydrogel.

[0106] In one embodiment, the cell population is suspended in an extracellular matrix, one or more extracellular matrix proteins, or a hydrogel. In one embodiment, the cell population is suspended in an extracellular matrix, one or more extracellular matrix proteins, or a hydrogel before being added to or filled into a first volume v1 (or first volume v1 and second volume v2). In one embodiment, the cell population is embedded in an extracellular matrix, one or more extracellular matrix proteins, or a hydrogel. In one embodiment, the cell population is sandwiched between a layer of a first extracellular matrix, one or more extracellular matrix proteins, or a hydrogel in volume v1 and a layer of a second extracellular matrix, one or more extracellular matrix proteins, or a hydrogel in volume v2.

[0107] The methods of the present disclosure may further include polymerizing a liquid extracellular matrix, one or more liquid extracellular matrix proteins, or liquid hydrogel (after being added to or filled into a first volume v1, or first volume v1 and second volume v2). Polymerizing the liquid extracellular matrix, one or more liquid extracellular matrix proteins, or liquid hydrogel (after being added to or filled into a first volume v1, or first volume v1 and second volume v2) may include incubating the liquid extracellular matrix, one or more liquid extracellular matrix proteins, or liquid hydrogel at a temperature suitable for polymerization.

[0108] A cell population can be exposed to cell culture medium when a liquid extracellular matrix, one or more liquid extracellular matrix proteins, or liquid hydrogel is added to or filled into a first volume v1, or a first volume v1 and a second volume v2, and optionally polymerized. In one embodiment, the cell population can be seeded on the polymerized extracellular matrix, one or more extracellular matrix proteins, or hydrogel and thus directly exposed to cell culture medium. In one embodiment, the cell population can be seeded or embedded in the polymerized extracellular matrix, one or more extracellular matrix proteins, or hydrogel and thus indirectly exposed to cell culture medium by diffusion or the like.

[0109] In one embodiment, the cell culture apparatus 1 comprises polymerized extracellular matrix up to the level of a first ledge 30, one or more extracellular matrix proteins or hydrogels (and a population of cells on or within them), and the cell culture medium is added to a second volume v2 surrounded by a second sidewall segment 25, and optionally to a third volume v3 surrounded by a third sidewall segment 35.

[0110] In one embodiment, the cell culture apparatus 1 includes polymerized extracellular matrix up to the level of a first ledge 30, one or more extracellular matrix proteins or hydrogels (and a population of cells on or within them), and a second volume v2 surrounded by a second sidewall segment 25 is filled with cell culture medium. In such an embodiment, the volume of the extracellular matrix, one or more extracellular matrix proteins or hydrogels (in the first volume v1) is equal to the volume of the cell culture medium (in the second volume v2).

[0111] Therefore, after sufficient exposure to the culture environment (e.g., extracellular matrix or cell culture medium), a cell population may grow or form one or more multicellular aggregates or organoids. Sufficient exposure time is typically measured in days or weeks, such as 3 days, 5 days, or 1 week, 2 weeks, 3 weeks or more.

[0112] The cell culture media are well-known and readily available, including organoid media commercialized by STEMCELL Technologies, such as culture medium products marketed under the brands IntestiCult®, HepatiCult®, PneumaCult®, PancreaCult®, and STEMdiff®. In some embodiments, different cell culture reagents may be added (or filled) to a second volume v2, or to a second volume v2 and a third volume v3, instead of cell culture media. Non-limiting examples may include digestive enzymes, or other reagents that may be used for downstream analysis of cell culture, such as reagents that may be required for staining operations, viability assessment, etc.

[0113] In one embodiment, a cell population may be treated with one or more test compounds and one or more control compounds (which may be diluted in a suitable culture medium) to evaluate the toxicity or reactivity to a candidate compound(s). In one embodiment, the one or more test compounds and one or more control compounds are each contained in the cell culture medium to which the cell population is (directly or indirectly) exposed in each well of the cell culture apparatus of the present disclosure.

[0114] Therefore, cells cultured using the cell culture apparatus 1 described herein according to the method described above may have several advantages over conventional approaches, particularly when forming or assaying multicellular aggregates or organoids. In fact, by controlling the shape and height of the polymerized extracellular matrix (or ECM protein, or hydrogel), variability in culture performance due to mass transfer rates can be limited or eliminated. As a result, three-dimensional cell assemblies or constructs, such as organoids, formed / induced therein may be more uniform in terms of size and / or function compared to the current state of dome approaches. Furthermore, three-dimensional cell assemblies or constructs, such as organoids, formed / induced in the cell culture apparatus of this disclosure may also exhibit improved seeding efficiency, proliferation, and / or function.

[0115] Furthermore, in the case of temperature-dependent gelling substrates, substrate proteins, or hydrogels, the shape and position of the substrate are constrained by the shape of the apparatus in the unpolymerized state, so the cell culture apparatus of this disclosure may reduce or eliminate the need for preheating. Typically, plates are preheated so that the substrate polymerizes more quickly in its original position, thereby attempting to reduce the opportunity for domes or droplets to spread, move, or deform before polymerization, which would increase the variability of the culture.

[0116] Furthermore, performing this method (using cell culture device 1) enhances control over substrate and cell deposition / localization, which can facilitate automation of imaging and analytical operations.

[0117] In one embodiment, an automated method for preparing and seeding in multiple wells (of the cell culture apparatus described herein) may rely on an automated liquid handler, as is known in the art.

[0118] In one embodiment, an automated method for assaying cell cultures in multiple wells (of the cell culture apparatus described herein) may depend on an automated imager mounted on a gantry or the like. Conversely, an automated method for assaying cell cultures in multiple wells (of the cell culture apparatus described herein) may depend on a fixed imager and a movable stage on which the cell culture apparatus of this disclosure is mounted.

[0119] The following non-limiting examples illustrate this disclosure. [Examples]

[0120] Example 1: Optimization of extracellular matrix depth (height of the first sidewall portion) The cell culture apparatus of this disclosure was manufactured by injection molding or 3D printing. In the case of 3D printing, the base and well definition members were formed using an Ultimaker S5 printer with ToughPLA filament, and then the two components were connected to each other. In some cases, such as when 3D printed, the cell culture apparatus of this disclosure was sterilized by UV irradiation before use.

[0121] In this embodiment, the effect of changing the height of the first side wall portion was investigated, and wells with first side wall portion heights of 0.75 mm, 1 mm, 1.25 mm, and 1.5 mm were manufactured.

[0122] Human liver organoids and human colon organoids were generated using HepatiCult® OGM (STEMCELL Technologies) or IntestiCult® OGM (STEMCELL Technologies), respectively, according to the manufacturer's recommendations. On the day of the experiment, the organoids were dissociated into small clumps according to the manufacturer's instructions and suspended in Matrigel® with 100% or 50% reduced growth factors. The cell suspension was added to the first or second ledge of each cell culture apparatus design, or applied as a dome on the bottom wall of a standard 24-well plate. After polymerization of the extracellular matrix, 500 μL of either HepatiCult® OGM (STEMCELL Technologies) or IntestiCult® OGM (STEMCELL Technologies) was added to each well.

[0123] Each well was imaged on days 3, 5, and 7 using tiling with a 5x wide-field Zeiss microscope and stitched together into a total well image. The total well images were analyzed using a Fiji macro to output the average cross-sectional area of ​​all organoids in the culture well.

[0124] Growth of liver and colon organoids in the cell culture apparatus of this disclosure is generally superior to that of standard dome culture methods ("conventional"), and the 0.75 mm, 1 mm, and 1.25 mm embodiments were superior to the 1.5 mm embodiment (Figures 5A and 5B).

[0125] Example 2: Characterization of organoid growth in the cell culture apparatus of the present disclosure Human intestinal organoids were maintained and dissociated in IntestiCult® OGM (STEMCELL Technologies) as recommended by the manufacturer. The resulting cell aggregate suspension was mixed with a Matrigel solution containing 50% reduced growth factors, and 50 μl of this mixture was filled into a volume enclosed by the first sidewall portion of the 24-well cell culture apparatus of this disclosure, or deposited as a dome on a conventional flat-bottomed 24-well plate. After polymerization of the extracellular matrix, 500 μL of IntestiCult® OGM (STEMCELL Technologies) was added to each well.

[0126] To evaluate growth, each well was imaged and analyzed in the same manner as in Example 1. The cell culture apparatus of this disclosure shows statistically greater growth in terms of cross-sectional area compared to conventional dome culture (Figure 6A).

[0127] To evaluate the seeding efficiency, organoids on day 7 were imaged and analyzed in the same manner as in Example 1. The number of seeded clumps at D0 was divided by the number of organoids with a diameter exceeding 100 μm that were counted during the culture on day 7. The cell culture apparatus of this disclosure exhibits statistically higher plating efficiency compared to conventional dome culture (Figure 6B).

[0128] To evaluate the size distribution of organoids, organoids were imaged and analyzed on day 7, as in Examples 1 and 2. The cell culture apparatus of the present disclosure generally shows reduced variability in the size distribution as measured by the average cross-sectional area per culture (Figure 6C). In contrast to organoids cultured in dome cultures, organoids cultured in the cell culture apparatus of the present disclosure did not appear to proliferate / grow into extremely large organoids.

[0129] Example 3: Evaluation of organoid functionality in the cell culture apparatus of the present disclosure Human intestinal organoids were dissected and seeded basically as described in Example 2. Forskolin (Orkambi) was used in the organoids. TMThe reactivity to gefitinib (data not shown) was evaluated. Organoids deposited as domes in a 24-well plate cell culture apparatus of this disclosure, or as domes in a standard 24-well plate, were exposed to 5 μM forskolin or DMSO for approximately 90 minutes. At various time points, each well was imaged and analyzed as described in Examples 1 and 2 to determine the mean cross-sectional area of ​​the organoids in culture.

[0130] Human intestinal organoids exposed to forskolin showed slightly improved swelling in the cell culture apparatus of this disclosure compared to standard dome culture. In particular, organoids treated in the cell culture apparatus of this disclosure showed a significantly reduced standard deviation compared to standard dome culture (Figures 7A and 7B). Dose-response experiments were performed on human intestinal organoids exposed to different doses of forskolin in either the 96-well plate cell culture apparatus of this disclosure or standard dome culture. Organoids evaluated in the cell culture apparatus of this disclosure showed a significantly stronger response to forskolin than organoids evaluated in standard dome culture (Figure 7C).

[0131] Example 4: Retention of extracellular matrix in the cell culture apparatus of the present disclosure Human liver organoids were dissociated essentially as described in Example 1. The resulting cell aggregate suspensions were mixed with Matrigel solutions containing 100%, 75%, 50%, 20%, and 10% reduced growth factors, and 50 μl of each mixture was packed into a volume enclosed by the first sidewall portion of the 24-well cell culture apparatus of this disclosure, or deposited as a dome on a conventional flat-bottom 24-well plate. After polymerization of the extracellular matrix, 500 μL of HepatiCult® OGM (STEMCELL Technologies) was added to each well, and a complete medium change was performed every 2-3 days. The cultures were imaged on days 4 and 7 using tiling with a 5x wide-field Zeiss microscope and stitched together into a full-well image.

[0132] The cell culture apparatus of this disclosure ("Improved") can support the deposition and gelation of Matrigel at lower concentrations / higher dilutions than can be successfully formed by dome culture (Figures 8A and 8B). Furthermore, in the case of highly diluted Matrigel, the cell culture apparatus of this disclosure supports larger organoid growth than conventional dome culture and exhibits less (in most cases none) volume disturbance and displacement of the gelled substrate than dome culture (Figures 8A and 8B).

[0133] Example 5: Quantification of evaporation levels from the cell culture apparatus of the present disclosure Standard 24-well and 96-well culture plates, or 24-well and 96-well cell culture devices of the present disclosure with a more fitted lid designed to reduce the gap between the underside of the lid and the lid support surface of the plate, were filled with 500 μL and 200 μL of distilled water colored with red dye, respectively. Daily absorbance was measured at 504 nm over 7 days, and the concentration of the red dye was correlated with the absorbance according to the Lambert-Beer law. "Corner" wells corresponded to each of the four corners. "Edge" wells corresponded to the outer wells that were not corners. "Internal" wells represented all of the remaining wells.

[0134] Figure 8 shows that, for each plate format (24-well or 96-well), the cell culture apparatus of this disclosure exhibits significantly lower evaporation rates between corner wells and edge wells compared to the corresponding standard culture plates.

[0135] The exemplary embodiments described herein are for illustrative purposes only and are not limiting. Many modifications are possible with respect to configuration, details, and sequence of operations. Rather, the present invention is intended to encompass all such modifications within its scope, as defined by the claims, and this should be given a broad interpretation consistent with the entire description.

Claims

1. A cell culture device, a) A well having a bottom wall and an opening, b) A first sidewall segment connected to the bottom wall and extending away from the bottom wall, wherein the first sidewall segment and the bottom wall define a first volume boundary for holding a liquid and / or polymerizable liquid, c) A second sidewall segment connected to the first sidewall segment by a first ledge, the plane of the second sidewall segment and the first ledge defining a second volume boundary for holding a liquid and / or polymerizable liquid, A cell culture apparatus in which the height of the first side wall segment is greater than the height of the second side wall segment.

2. The cell culture apparatus according to claim 1, wherein the first volume is equal to the second volume with an error of ±10%.

3. The cell culture apparatus according to claim 1 or 2, wherein the height of the first side wall segment is approximately 0.1 mm to 10 mm, preferably 0.5 mm to 5 mm.

4. The cell culture apparatus according to any one of claims 1 to 3, wherein the height of the second side wall segment is approximately 0.1 mm to 10 mm, preferably 0.2 mm to 4 mm.

5. The cell culture apparatus according to any one of claims 1 to 4, wherein the first sidewall segment forms an obtuse angle with the bottom wall, and / or the second sidewall segment forms an obtuse angle with the first ledge.

6. The cell culture apparatus according to any one of claims 1 to 5, wherein the width of the first ledge is approximately 0.1 mm to approximately 5 mm.

7. The cell culture apparatus according to any one of claims 1 to 6, wherein one or both of the first side wall segment and the second side wall segment are circular or substantially circular in the plane of the bottom wall.

8. The cell culture apparatus according to claim 7, wherein the diameter of the first side wall segment at any position along its height is smaller than the diameter of the second side wall segment at any position along its height.

9. The cell culture apparatus according to claim 8, wherein the diameter of the first side wall portion is approximately 2 mm to 10 mm, approximately 2.5 mm to 9 mm, or approximately 3 mm to 8 mm.

10. The cell culture apparatus according to claim 8 or 9, wherein the diameter of the second side wall portion is approximately 3 mm to 15 mm, approximately 3.5 mm to 14 mm, or approximately 4 mm to 13 mm.

11. The cell culture apparatus according to any one of claims 1 to 10, further comprising a third sidewall segment connected to the second sidewall segment by a second ledge, wherein the planes of the third sidewall segment and the second ledge define a third volume boundary for holding a liquid and / or polymerizable liquid.

12. The cell culture apparatus according to claim 11, wherein the third side wall portion extends from the second ledge to the opening.

13. The cell culture apparatus according to claim 11 or 12, wherein the height of the third side wall segment is greater than the height of the second side wall segment, and the volume of the third is greater than the volume of the second.

14. The cell culture apparatus according to any one of claims 11 to 13, wherein the height of the third side wall segment is approximately 1 to 15 mm or approximately 2 to 10 mm.

15. The cell culture apparatus according to any one of claims 11 to 14, wherein the second ledge and the third side wall segment form an obtuse angle.

16. The cell culture apparatus according to any one of claims 11 to 15, wherein the width of the second ledge is approximately 0.2 mm to approximately 5 mm.

17. The cell culture apparatus according to any one of claims 11 to 16, wherein the third side wall portion is substantially circular in the plane of the bottom wall, and the diameter of the third side wall segment at any position along its height is greater than the diameter of the second side wall segment at any position along its height.

18. It further includes multiple wells, each well having at least a) Bottom wall and opening, b) A first sidewall segment connected to the bottom wall and extending away from the bottom wall, wherein the first sidewall segment and the bottom wall define a first volume boundary for holding a liquid and / or polymerizable liquid, c) A second sidewall segment connected to the first sidewall segment by a first ledge, the plane of the second sidewall segment and the first ledge defining a second volume boundary for holding a liquid and / or polymerizable liquid, The cell culture apparatus according to any one of claims 1 to 17, wherein the height of the first side wall segment is greater than the height of the second side wall segment.

19. A method for culturing cells, comprising seeding a population of cells into a well of a cell culture apparatus according to any one of claims 1 to 18.

20. The method according to claim 19, further comprising adding a liquid extracellular matrix, one or more liquid extracellular matrix proteins, or a liquid hydrogel to the first volume enclosed by the first side wall segment of the cell culture apparatus.

21. The method according to claim 20, further comprising polymerizing the liquid extracellular matrix, the one or more liquid extracellular matrix proteins, or the liquid hydrogel.

22. The method according to claim 21, wherein the cell population is suspended and / or embedded in the extracellular matrix, one or more extracellular matrix proteins, or the hydrogel.

23. The method according to claim 22, further comprising exposing the cell population to the cell culture medium by adding the cell culture medium to the second volume surrounded by the second side wall segments.

24. The method according to claim 23, wherein the volume of the extracellular matrix, the one or more extracellular matrix proteins, or the hydrogel is equal to the volume of the cell culture medium.

25. The method according to claim 23 or 24, further comprising generating a multicellular aggregate or organoid from the aforementioned cell population.

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