Open-well type microcavity plate

Abstract

We provide cell culture devices. [Solution] The cell culture device comprises a frame 102 having an open well 150 disposed within it and a fluid inlet area 105 communicating with the open well. The open well has an upper opening, a bottom plate that constitutes a microcavity substrate 160 and defines the main surface, and one or more side walls 120 extending from the bottom plate to the upper opening. This microcavity plate 100 comprises a plurality of microcavities, each microcavity designed so that cells cultured in the well form spheroids. This cell culture device may be a storage open-well type microcavity plate used for spheroid cell culture.

Description

Description of Related Applications

[0001] This application claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 63 / 116,280, filed November 20, 2020, the contents of which are relied upon and are hereby incorporated in their entirety by reference.

Technical Field

[0002] The present disclosure broadly relates to cell culture devices and methods. In particular, the present disclosure relates to an open-well type microcavity plate for use in cell culture.

Background Art

[0003] Cells cultured in a three-dimensional (3D) cell culture environment exhibit more in vivo-like functions than cells cultured in a two-dimensional (2D) environment as a monolayer. In a 2D cell culture system, cells adhere to the substrate on which they are cultured. In contrast, when cells are grown in a 3D system, they interact with each other rather than adhere to the substrate to form a 3D cell culture or spheroid.

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, growing 3D cell cultures in conventional cell culture systems presents challenges. One difficulty is maintaining consistent size and culture environment for spheroids grown in separate wells of the cell culture system. For example, seeding density and growth time will affect reproducibility across systems or between wells within a given system. As the density of cells grown in the cell culture system increases, maintaining the cells requires larger volumes of cell culture medium or more frequent changes of the cell culture medium. Increased frequency of medium changes will cause the medium height to rise above the cells being cultured, undesirably reducing the rate of gas exchange between cells through the medium. Furthermore, conventional methods of growing cells at high density as spheroid clusters in wave bags, spinners, and shakers can exhibit inconsistency in growth, and may tend to split spheroids into smaller clusters. Spheroid clusters are often fragmented or disturbed during medium changes, so there are also challenges when conventional cell culture plates or tissue culture plates are used to grow spheroids at high density. [Means for solving the problem]

[0005] Embodiments of this disclosure provide a microcavity plate for bulk spheroid production that facilitates culture exchange. A fluid inlet area within the microcavity plate provides a place to insert the tip of a pipette during culture exchange. By providing a pipette tip area, the embodiments described herein solve the problem of existing equipment where the tip is inserted into the culture area and disturbs or displaces the spheroids. The fluid inlet area also allows for the introduction of fluid into the plate while minimizing sloshing and turbulence. The fluid inlet area can also function as a fluid outlet area. The microcavity plate comprises an open well with a microcavity base portion having a shallow microcavity for growing spheroids. When the tip of the pipette is inserted into the fluid inlet area and fluid is introduced into the microcavity plate, turbulence from the incoming fluid is minimized, and the spheroids are not displaced or disturbed from the microcavity by the fluid or the pipette tip.

[0006] By providing a microcavity substrate within an open well, the embodiments of this disclosure enable the simultaneous cultivation of spheroids in the same environment from the start of the culture process. By simultaneously culturing spheroids in the same environment, disruption to the spheroid clusters due to medium changes is minimized while ensuring consistency in size and growth, thereby solving the problems of existing conventional equipment.

[0007] In one embodiment, the cell culture device comprises a frame having an open well disposed therein and a fluid inlet area communicating with the open well. The open well has an upper opening, a bottom plate constituting a microcavity substrate and defining a main surface, and one or more side walls extending from the bottom plate to the upper opening.

[0008] In some embodiments, the fluid inlet area includes a sidewall surface of one or more sidewalls. In some embodiments, the sidewall surface slopes from the upper outer portion of the sidewall to the bottom inner portion of the sidewall along the length of the sidewall. In some embodiments, the upper outer portion is at the same level as the upper opening. In some embodiments, the bottom inner portion is at the same level as the main surface and communicates with the main surface.

[0009] In some embodiments, the fluid inlet area has a notch located within one or more side walls. In some embodiments, the notch includes a tetrahedral notch at the center of the side wall. In some embodiments, the edge of the tetrahedral notch slopes from the upper outer portion of the side wall to the bottom inner portion of the side wall. In some embodiments, the upper outer portion is at the same level as the upper opening. In some embodiments, the bottom inner portion is at the same level as the main surface and communicates with the main surface.

[0010] In some embodiments, the fluid inlet area has a notch located at the corner of the open well, where the first sidewall of one or more sidewalls is joined at a right angle to the second sidewall of one or more sidewalls. In some embodiments, the notch includes a tetrahedral notch at the corner of the open well. In some embodiments, the edge of the tetrahedral notch slopes from the upper outer portion of the corner to the bottom inner portion of the corner. In some embodiments, the upper outer portion of the corner is at the same level as the upper opening. In some embodiments, the bottom inner portion of the corner is at the same level as the main surface and communicates with the main surface.

[0011] In some embodiments, the fluid inlet area includes a ledge located within one or more side walls. In some embodiments, the ledge is a grooved passage. In some embodiments, the ledge slopes from the top of the first end of the side wall to the bottom of the second end of the side wall. In some embodiments, the bottom is at the same level as the main surface and communicates with the main surface. In some embodiments, the top is at the same level as the top opening.

[0012] In some embodiments, the fluid inlet area is the fluid outlet area.

[0013] In some embodiments, the cell culture device further includes a baffle. In some embodiments, the baffle is located in an open well between the main surface and the upper opening. In some embodiments, the baffle includes a plurality of baffle segments, each baffle segment extending from one end of the open well to the opposite end of the open well. In some embodiments, at least one of the plurality of baffle segments is perpendicular to the other baffle segments. In some embodiments, the first baffle segment is located in the open well along the length of the side wall, adjacent to the fluid inlet area.

[0014] In some embodiments, the microcavity substrate comprises a plurality of microcavities. In some embodiments, the plurality of microcavities are arranged in at least one row. In some embodiments, the plurality of microcavities are arranged in a hexagonal close-packed pattern.

[0015] In some embodiments, each microcavity of a plurality of microcavities has an upper opening, a bottom, and a microcavity sidewall surface extending from the upper opening to the bottom of the microcavity. In some embodiments, the upper opening of the microcavity is coplanar with the main surface, and the bottom of the microcavity is located below the main surface. In some embodiments, each microcavity has a rounded bottom. In some embodiments, the width of the upper opening of each microcavity is 500 μm to 5 mm. In some embodiments, the depth of each microcavity of a plurality of microcavities is 500 μm to 6 mm.

[0016] In some embodiments, each microcavity is non-adherent to cells. In some embodiments, the internal surface of each microcavity is coated with a very low-adhesion material. In some embodiments, each microcavity is designed so that cells cultured in the well form spheroids.

[0017] In some embodiments, one or more sidewalls define a storage area on the microcavity substrate. In some embodiments, one or more sidewalls have a height of 0.780 inches (approximately 20 mm).

[0018] In some embodiments, the inner surface of the open well is non-adherent to cells. In some embodiments, the inner surface of the open well includes a non-adherent surface coating made from perfluoropolymers, olefins, agarose, nonionic hydrogels, polyethers, polyols, polymers that inhibit cell adhesion, or combinations thereof. In some embodiments, the non-adherent surface coating includes an extremely low adhesion (ULA) surface coating.

[0019] In some embodiments, the frame, one or more sidewalls, or a combination thereof is formed from polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polymethylpentene, polycarbonate, polymethylmethacrylate, styrene-ethylene-butadiene-styrene, silicone rubber or copolymer, ethylene vinyl acetate, polysulfone, polytetrafluoroethylene, poly(styrene-butadiene-styrene), or a combination thereof.

[0020] In some embodiments, the microcavity substrate is formed from polydimethylsiloxane (PDMS), polymethylpentene, (poly)4-methylpentene (PMP), polyethylene (PE), polystyrene (PS), polypropylene, polyethylene terephthalate, polycarbonate, polymethylmethacrylate, styrene-ethylene-butadiene-styrene, silicone rubber or copolymer, ethylene vinyl acetate, polysulfone, polytetrafluoroethylene, poly(styrene-butadiene-styrene), or a combination thereof.

[0021] In some embodiments, the cell culture device is a storage-release well type microcavity plate.

Brief Description of the Drawings

[0022] [Figure 1] View from above of an embodiment of the cell culture device [Figure 2] Vertical cross-sectional view of the cell culture device shown in FIG. 1 [Figure 3] View from above of an embodiment of the cell culture device [Figure 4] Enlarged view of the features of the cell culture device shown in FIG. 3 [Figure 5] View from above of an embodiment of the cell culture device [Figure 6] Enlarged view of the features of the cell culture device shown in FIG. 5 [Figure 7] View from above of an embodiment of the cell culture device [Figure 8] Enlarged view of the features of the cell culture device shown in FIG. 7 [Figure 9] Enlarged view of an embodiment of the microcavity substrate [Figure 10] Enlarged view of an embodiment of the microcavity substrate [Figure 11] Enlarged view of an embodiment of the microcavity substrate

Mode for Carrying Out the Invention

[0023] Cell responses in 3D cell cultures such as 3D spheroids and organoids (hereinafter referred to as spheroids) are more similar to in vivo behavior than cell responses in 2D cell cultures where cells are cultured in a monolayer. The additional dimensionality of 3D cultures is thought to bring about differences in cell responses. This is because the additional dimensionality affects the spatial configuration of cell surface receptors involved in interactions with surrounding cells, imposes physical constraints on the cells, thereby acting on signal transduction from the outside to the inside of the cells, and ultimately affecting gene expression and cell behavior. However, conventional culture devices for generating 3D cell cultures or spheroids have defects in medium exchange. Spheroids may be pushed away or disrupted by the tip of a pipette or by the introduced fluid when the medium is exchanged.

[0024] Furthermore, in contrast to two-dimensional cell cultures in which cells form a monolayer on the surface, the formation of three-dimensional (3D) cell aggregates such as spheroids increases the density of cells grown in the cell culture device. As the cell density increases, the demand for nutrients for the cells cultured in the device also increases. Therefore, during the bulk culture of spheroids, medium exchange is required more frequently.

[0025] In some embodiments, a cell culture device is provided comprising a frame in which an open well is disposed. The open well has an upper opening, a microcavity substrate bottom, and one or more side walls extending from the bottom to the upper opening. The cell culture device further comprises a fluid inlet area.

[0026] The embodiments described herein provide a storage microcavity plate having a separate area from the microcavity culture area for adding or removing fluid. As described herein, the storage plate may be referred to as an "open-well" plate. In the embodiments, the open-well plate is made from a wavy microcavity substrate in which both the upper and lower surfaces of the microcavity substrate are undulating.

[0027] The microcavity plate according to the embodiments described herein has a fluid inlet area. The fluid inlet area is sized to receive the tip of a pipette for fluid introduction or aspiration. The fluid inlet area is in fluid communication with the microcavity substrate. In the embodiments, the fluid inlet area may also be the fluid outlet area.

[0028] In some embodiments, the fluid inlet area has a bottom that is spaced away from the microcavity substrate, such as being spaced at a higher height or relative height. In some embodiments, the fluid inlet area is spaced away from the microcavity substrate to divert the fluid being dispensed from the pipette, thus avoiding breaking or disturbing the spheroid.

[0029] The embodiments of open-well microcavity plates described herein may be covered with a standard microplate lid to reduce the possibility of contamination of the culture. The lid may be lifted completely away from the culture or simply moved aside to expose the area for pipetting.

[0030] Figure 1 shows a top-down view of an embodiment of a cell culture device, such as a microcavity plate 100. Figure 2 shows an enlarged vertical cross-sectional view of the microcavity plate 100 of Figure 1. This cell culture device may include a frame 102 having an open well 150 and a fluid inlet area 105 communicating with the open well 150. The open well 150 has an upper opening 155, a bottom plate 162 that constitutes a microcavity base 160 defining a main surface 161, and one or more side walls 120, 121, 122, 123 extending from the bottom plate 162 to the upper opening 155. A skirt 170 may be placed around the cell culture device to provide stability to the device, for example.

[0031] As shown in Figures 1 and 2, the microcavity plate 100 has a long fluid inlet / outlet area 105, which extends along the width of the growth surface area. The fluid inlet area 105 includes a surface 128 of sidewall 120, one of one or more sidewalls 120, 121, 122, 123. The surface 128 of sidewall 120 slopes from the upper outer portion 124 of this sidewall to the bottom inner portion 126 of this sidewall along the length of sidewall 120. The upper outer portion 124 is at the same level as the upper opening 155. The bottom inner portion 126 is at the same level as the main surface 161 and communicates with the main surface 161. The main surface 161 is the cell growth area defined by the microcavity substrate 160. Since the width 125 at the top of the side wall 120 is narrower than the width 127 at the bottom of the side wall 120, the side wall 120 in which the fluid inlet area is located is sometimes described as being inclined.

[0032] The cell culture device may further include a baffle 190. The baffle 190 may be constructed to fit into an open well and may include multiple baffle segments, such as baffle segment 191, baffle segment 193, and baffle segment 195. As shown in Figure 1, baffle segment 191 may extend from side wall 120 to the opposite side wall 122 and be positioned along the central length of the open well. Baffle segments 193 and 195 may extend along the width of the open well from side wall 121 to the opposite side wall 123 and be positioned perpendicular to baffle segment 191. Baffle segment 193 may be positioned along the central width of the open well. Baffle segment 195 may extend along the first side wall 120 and be positioned parallel to baffle segment 193 at the end of baffle segment 191 adjacent to the first side wall 120. The baffle segment 195 forms a single wall in the fluid inlet / outlet area, which can help hold the pipette tip in place. The user can insert the tip of a stripette (e.g., Corning® Coaster® Stripette® serological pipette) into the fluid inlet / outlet area to remove and distribute fluid during medium exchange.

[0033] Figure 3 shows a top-down view of an embodiment of the microcavity plate 200. Figure 4 shows a magnified view of the features of the fluid inlet area of ​​the microcavity plate 200 of Figure 3. As shown in Figures 3 and 4, the microcavity plate 200 has a long, angled storage or fluid inlet area 205. This long, angled storage or fluid inlet area 205 extends along the width of the growth surface area and tapers towards the edge.

[0034] The cell culture device may further include a baffle 290 designed to fit into an open well. The baffle 290 may include multiple baffle segments, such as baffle segment 291, baffle segment 293, and baffle segment 295. As shown in Figure 3, baffle segment 291 may extend from side wall 220 to the opposite side wall 222 and be positioned along the central length of the open well. Baffle segments 293 and 295 may extend along the width of the open well from side wall 221 to the opposite side wall 223 and be positioned perpendicular to baffle segment 291. Baffle segment 293 may be positioned along the central width of the open well. Baffle segment 295 may extend along the first side wall 220 and be positioned parallel to baffle segment 293 at the end of baffle segment 291 adjacent to the first side wall 220. The baffle segment 295 may constitute one wall of the space in the fluid inlet area 205, which can help restrain the tip of the strippet used for adding and removing fluid.

[0035] The fluid inlet region 205 includes a notch located within one or more side walls 220. The surface areas or faces 287, 288 of the notch on the side wall 220 opposite the baffle segment 295 are inclined toward each other, forming an edge 241. Thus, the notch 205 forms a tetrahedral notch at the center 229 of the side wall, where the edge 241 of the tetrahedral notch is inclined from the upper outer portion 224 of the side wall 220 to the bottom inner portion 226 of the side wall 220. The upper outer portion 224 may be at the same level as the upper opening. The bottom inner portion 226 may be at the same level as the main surface 261 and may communicate with the main surface 261. The main surface 261 is a cell proliferation region defined by the microcavity substrate 260.

[0036] Figure 5 shows a top-down view of an embodiment of the microcavity plate 300. Figure 6 shows a close-up view of the features of the fluid inlet area of ​​the microcavity plate 300 of Figure 5. As shown in Figures 5 and 6, the microcavity plate 300 has a triangular storage area or notch as a fluid inlet area 305. This notch or triangular storage area provides an area for the user to place the tip of a strippet for adding and removing fluid. The fluid inlet area 305 includes a triangular storage area or notch located at the corner of the open well 350, where a first side wall 320 of one or more side walls joins a second side wall 321 of one or more side walls at a right angle. The triangular storage area is entirely within the boundary of the plate structure or frame 302 outside the growth surface area, except for the bottom where it joins with the growth surface area defined by the microcavity substrate 360. The faces 387 and 388 of the side walls 320 and 321 that form the two outer sides of the triangle are inclined toward the region 336 that joins with the growth surface region. The faces 387 of the first side wall and 388 of the second side wall are inclined and form the edge 341 where they meet. Thus, the fluid inlet region 305 includes a tetrahedral notch at the corner of the open well 350, where the edge 341 of the tetrahedral notch is inclined from the upper outer portion 334 of the corner to the bottom inner portion 336 of the corner. The upper outer portion 334 of the corner is at the same level as the upper opening. The bottom inner portion 336 of the corner is at the same level as the main surface 361 and communicates with the main surface 361. The main surface 361 is defined by the microcavity substrate 360 ​​and is the cell growth region or cell growth surface.

[0037] The cell culture device may further include a baffle 390 designed to fit into an open well. The baffle 390 may include multiple baffle segments, such as baffle segment 391, baffle segment 393, and baffle segment 395. As shown in Figure 5, baffle segment 391 may extend from side wall 320 to the opposite side wall 322 and be positioned along the central length of the open well. Baffle segments 393 and 395 may extend along the width of the open well from side wall 321 to the opposite side wall 323 and be positioned perpendicular to baffle segment 391. Baffle segment 393 may be positioned along the central width of the open well. Baffle segment 395 may extend along the first side wall 320 and be positioned parallel to baffle segment 393 at the end of baffle segment 391 adjacent to the first side wall 320.

[0038] Figure 7 shows a top view of an embodiment of the microcavity plate 400. Figure 8 shows a close-up view of the features of the fluid inlet area 405 of the microcavity plate 400 of Figure 7. As shown in Figures 7 and 8, the microcavity plate 400 includes a fluid inlet area 405 that extends along the width of the growth surface area of ​​the open well 450. The fluid inlet area 405 includes a fluid ledge or groove 407 carved out from the periphery area of ​​the plate structure. The fluid inlet area 405 includes a ledge or grooved passage 407 located in one or more side walls 420, 421, 422, 423, in the side wall 420. The ledge 407 slopes from the top 485 of the first end 481 of the side wall 420 to the bottom of the second end 483 of the side wall 420. The bottom is at the same level as the main surface 461 and communicates with the main surface 461. The main surface 461 is a cell proliferation region defined by the microcavity substrate 460. The upper part 485 is at the same level as the upper opening. Thus, the fluid inlet region 405 is higher on the side furthest from the proliferation surface and decreases in width as the width moves diagonally, ending about 2 mm above the proliferation surface. The user can place the tip of a strippet on the top of the ledge 405 at the first end 481 of the side wall to add fluid, and place its tip on the bottom of the ledge 405 at the second end 483 of the side wall to remove fluid.

[0039] The cell culture device may further include a baffle 490 designed to fit into an open well. The baffle 490 may include multiple baffle segments, such as baffle segment 491, baffle segment 493, and baffle segment 495. As shown in Figure 7, baffle segment 491 may extend from side wall 420 to the opposite side wall 422 and be positioned along the central length of the open well. Baffle segments 493 and 495 may extend along the width of the open well from side wall 421 to the opposite side wall 423 and be positioned perpendicular to baffle segment 491. Baffle segment 493 may be positioned along the central width of the open well. Baffle segment 495 may extend along the first side wall 420 and be positioned parallel to baffle segment 493 at the end of baffle segment 491 adjacent to the first side wall 420.

[0040] In some embodiments, the cell culture apparatus may comprise a bottom plate or base and one or more side walls. In some embodiments, the cell culture apparatus described herein comprises a bottom plate defining a main surface, one or more side walls extending from the bottom plate defining a storage area, and a plurality of microcavities formed in the main surface. The bottom plate may be formed entirely or in part from a substrate having an array of microcavities that promote or induce spheroid growth. Each microcavity is coplanar with the main surface and defines an upper opening that opens into a storage area, and the top and bottom of microcavities located below the main surface. In contrast to conventional well plates, the plate described herein defines the storage area above the surface of the microcavities, thereby increasing the volume of cell culture medium to be used, and therefore reducing the frequency of medium changes. The storage plate described herein allows for the addition of excess medium beyond what would typically be used to fill the individual shallow wells of a microwell plate, and allows for fluid communication between cells cultured in different microcavities.

[0041] In some embodiments, one or more sidewalls extend further from the bottom plate (e.g., sidewall height) than in some commercially available cell culture devices, allowing the storage section to hold a larger volume of culture medium than usual. For opportunities to have a larger storage capacity, excess medium can be added to the storage section so that the spheroids do not have to depend solely on the amount of medium in each individual microcavity. Since the cell culture medium in the storage section is in communication with all microcavities within the storage section, nutrients and metabolites can be exchanged throughout the cell culture medium. Therefore, spheroids cultured in the embodiments of the microcavity plates described herein do not need to be fed (i.e., have their cell culture medium replaced) as frequently as spheroids growing in the wells of a standard microplate. When feeding is required, the cell culture medium can be added in the fluid inlet area to prevent the spheroids from being pushed out of the shallow wells by fluid movement.

[0042] In one embodiment, the storage open-well microcavity plate described herein may have deeper sidewalls (for example, deeper sidewalls than those of a standard well plate) to hold a larger volume of culture medium than usual. For example, the height of the cell culture device or microcavity plate may be approximately 0.780 inches (approximately 20 mm) compared to the height of a standard 96-well or 384-well plate of 0.560 inches (approximately 14 mm) (with a given dimensional tolerance of ±0.010 inches (approximately 0.25 mm)).

[0043] The microcavity substrate according to the embodiments described herein comprises a plurality of microcavities. Each microcavity may contain an internal cavity with a rounded bottom that is non-adherent to cells. Thus, the cell culture device described herein facilitates 3D cell culture by allowing cells seeded in the microcavities to self-organize or adhere to one another to form spheroids within each microcavity. The microcavities are shallow, and the cell culture medium can cover all of the spheroids in all the cavities at once, facilitating manual handling.

[0044] In one embodiment, the top surface of a microcavity may be recessed to a position close to the bottom of the side wall. Each microcavity can hold a small volume of culture medium. Each microcavity may have any suitable dimensions. For example, the diameter or width of an individual microcavity may range from about 500 micrometers to about 5 mm. The depth of an individual microcavity may range from about 500 micrometers to about 6 mm. An excess of culture medium may be added to the storage area so that the spheroids do not have to rely solely on the small amount of culture medium in each individual microcavity.

[0045] Figure 9 shows an enlarged view of an embodiment of a microcavity substrate having a pattern of microcavity arrays forming the bottom surface of an open well. The enlarged view of the microcavity substrate 900 includes a microcavity 910 or array of microwells. The structured surface of a cell culture apparatus having the microcavity or array of microwells described herein may define any number of microcavities, which may have any appropriate size and shape. The microcavities define the volume based on their size and shape. In many embodiments, one or more of the microcavities are symmetrical and / or rotationally symmetrical with respect to the longitudinal axis. In some embodiments, the longitudinal axes of one or more of the microcavities are parallel to each other. The microcavities may be spaced uniformly or non-uniformly. In some embodiments, the microcavities are spaced uniformly. One or more of the microcavities may have the same size and shape, or they may have different shapes and sizes.

[0046] In some embodiments, the microcavity substrate defining the microcavities comprises an array of hexagonal close-packed microcavities. Such a hexagonal close-packed density, or "honeycomb" microcavity morphology, combined with the micro-sized structure of the microcavities, enables the cultivation of many spheroids at once, allowing for bulk spheroid production. Figure 10 shows an image of an embodiment of such a substrate 1000, which has an array of hexagonal microcavities 1001. In one embodiment, such a packing density allows for the inclusion of approximately 12,588 wells with a diameter of 500 μm on a typical microplate working surface area of ​​approximately 4.5 inches × approximately 3 inches (approximately 11 cm × 7.6 cm). Figure 11 shows an embodiment of substrate 1100 in which cells (spheroids) 500 are being grown within microcavities 1101, and the array of microcavities has a hexagonal close-packed microcavity structure. In some embodiments, the cells within each microcavity 1101 form a single spheroid 500, as can be seen in the figure.

[0047] The microcavity plate according to the embodiments of this disclosure provides a uniform culture environment. All spheroids cultured in this microcavity plate are treated in the same way at the same time, thereby providing a uniform culture environment. In contrast, a typical plate with individual wells tends to have a non-uniform culture environment because it is difficult to distribute the same volume to each well, even with automated equipment.

[0048] In certain embodiments, the cell culture apparatus described herein includes a microcavity substrate as the bottom surface of the open well. The microcavity substrate comprises a plurality of microcavities. Each microcavity in the plurality of microcavities may be constructed such that cells cultured within the microcavity form a spheroid of a specified diameter. The microcavities may be of any size suitable for culturing spheroids or 3D cell cultures. In some embodiments, the width of the microcavities may range from about 500 micrometers to about 5 mm. In some embodiments, the depth of the microcavities may range from about 500 micrometers to about 6 mm. For example, in embodiments with larger microcavities, the microcavities overlap with the well size of the spheroid plate, thereby allowing organoids to grow in bulk culture.

[0049] In some embodiments, the cell culture device comprises 8 to about 10,000 microcavities (8, 16, 24, 32, 48, 64, 96, 128, 256, 384, 500, 600, 700, 800, 1000, 1536, 2000, 2400, 3200, 4000, 10000, or any range thereof). In some embodiments, multiple microcavities are arranged in at least one row. In some embodiments, the device comprises multiple rows of microcavities. In some embodiments, the microcavity substrate provides a structured surface defining multiple gas-permeable wells or microcavities. In some embodiments, the microcavities are in gaseous communication with the outside of the device through a gas-permeable material. In some embodiments, the structured surface defines multiple gas-permeable microcavities.

[0050] Depending on the initial polymer film thickness and process parameters, a surface with microwells having different bottom thicknesses is produced. In some embodiments, the polymer thickness at the bottom of the microwells directly affects oxygen permeability. The thinner the bottom of the microwells, the better the oxygen supply to the cells within the microwells. The above manufacturing method provides a surface with microwells having higher oxygen permeability.

[0051] In some embodiments, each of the multiple microcavities defines a top opening, a microwell bottom, and a microcavity sidewall extending from the top opening to the microwell bottom. The microcavity opening or top opening may have any suitable shape. For example, the opening may be circular, hexagonal, etc. In some embodiments, the microcavity bottom constitutes a rounded bottom. In some embodiments, the bottom of each microcavity is rounded (e.g., hemispherical), the microcavity sidewalls increase in diameter from the bottom to the top of the microcavity, and the boundaries between adjacent microcavities are rounded.

[0052] In some embodiments, the shape of the microcavity is transitioned to mitigate the problem of air escaping when a liquid is introduced into the microcavity. In some embodiments, the bottom (or bottom portion) of a microcavity with a circular cross-section may be optimal for spheroid formation but would be problematic for air escaping without forming a pocket. To mitigate this problem, microcavities may be formed with a circular well bottom cross-section and a non-circular (e.g., triangular, square, rectangular, pentagonal, hexagonal, etc.) top opening. In such embodiments, the sidewalls transition from the non-circular (e.g., polygonal) top opening to the circular bottom of the microcavity. In some embodiments, the transition is gradual so as not to introduce any interfering, jagged, or horizontally appearing sidewall features of the microcavity that could cause "hanging up" of air bubbles escaping from the microcavity when a liquid is introduced into the microcavity. In some embodiments, the corners in the sidewalls of the microcavity, resulting from the non-circular (e.g., polygonal) shape of the transitioning wall and the upper opening, provide pathways for liquid entry and / or air escape.

[0053] The microcavity substrate may be formed from the same or similar material by a method for manufacturing the rest of the plate. In some embodiments, the microcavity substrate may be molded or formed separately from the rest of the plate and then joined by any other method such as thermal bonding, ultrasonic welding, or plastic bonding. The material of the microcavity substrate structure may be made from plastic polymers, copolymers, or polymer blends. Non-limiting examples include silicone rubber, polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polymethylpentene, polycarbonate, polymethyl methacrylate, styrene-ethylene-butadiene-styrene, other such polymers, or combinations thereof. Any suitable construction method may be used to form the microcavity substrate, non-limiting examples of which include injection molding, thermoforming, 3D printing, or any other method suitable for forming plastic parts.

[0054] The microcavity plates according to the embodiments described herein may be formed from any suitable material. The constituent material may be made from a plastic polymer, copolymer, or polymer blend. Non-limiting examples include silicone rubber, polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polymethylpentene, polycarbonate, polymethyl methacrylate, styrene-ethylene-butadiene-styrene, other such polymers, or combinations thereof. Any suitable method of formation may be used to form the microcavity substrate. Non-limiting examples include injection molding, thermoforming, 3D printing, or any other method suitable for forming plastic parts.

[0055] In some embodiments, gas-permeable / liquid-impermeable materials are used in the construction of the cell culture devices described herein. Any suitable gas-permeable / liquid-impermeable material may be used in the embodiments described herein. Non-limiting examples of gas-permeable / liquid-impermeable materials include polystyrene, polycarbonate, ethylene vinyl acetate, polysulfone, polymethylpentene (PMP), polytetrafluoroethylene (PTFE) or compatible fluoropolymers, silicone rubber or copolymers, poly(styrene-butadiene-styrene), or polyolefins such as polyethylene or polypropylene, or combinations thereof. The microcavity substrate may be formed from any suitable material having appropriate gas permeability over at least a portion of the well. Suitable microcavity substrates, though not limited to specific examples, include polydimethylsiloxane (PDMS), polymethylpentene, (poly)4-methylpentene (PMP), polyethylene (PE), polystyrene (PS), polypropylene, polyethylene terephthalate, polycarbonate, polymethyl methacrylate, styrene-ethylene-butadiene-styrene, silicone rubber or copolymer, ethylene vinyl acetate, polysulfone, polytetrafluoroethylene, poly(styrene-butadiene-styrene), or combinations thereof. Such materials enable effective gas exchange between the cell culture area of ​​the microcavity and the external environment, allowing the entry of oxygen and other gases while preventing the passage of liquids and contaminants.

[0056] In some embodiments, the thickness of the microcavity substrate is adjusted to optimize gas exchange. The thickness of the microcavity substrate depends on the constituent material. In some embodiments, the thickness of the bottom of the microcavity is between 10 μm and 75 μm (e.g., 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm, 75 μm, and any range in between). In some embodiments, the microcavity has a capacity of 2000 cc / m³. 2The microcavity substrate has an oxygen permeability of 3000 cc / m² or more through a gas-permeable polymer material. In some embodiments, the microcavity has an oxygen permeability of 3000 cc / m². 2 It has a gas permeability through the substrate of 5000 cc / m² or more. In some embodiments, the microcavity has a gas permeability of 5000 cc / m². 2 It has a gas permeability of more than / day through the substrate.

[0057] The cell culture devices described herein enable the generation and culture of 3D cell aggregates. Cells cultured in three dimensions, such as spheroids, can exhibit more in vivo-like functions than cells cultured in two dimensions as a monolayer. In 2D cell culture systems, cells can adhere to the substrate on which they are cultured. However, when cells are grown in three dimensions, such as in spheroids, they interact with each other rather than adhere to the substrate. Cells cultured in three dimensions closely resemble in vivo tissues in terms of cell communication and the development of the extracellular matrix. Therefore, spheroids provide an excellent model for cell migration, differentiation, survival, and proliferation, and thus offer a better system for research, diagnostics, and efficacy, pharmacology, and toxicity testing.

[0058] In some embodiments, a microcavity substrate forms part of a microcavity plate for growing cells or spheroids. The microcavity is configured and arranged to provide an environment that promotes spheroid formation during culture. That is, in some embodiments, the microcavity has a spheroid-inducing structure. For example, a microcavity in which cells are grown may be non-adherent to the cells within the microcavity so that the cells bind to each other and form spheres. The spheroids expand to the size limit imposed by the structure of the microcavity. In some embodiments, the cell culture substrate within the device is non-adherent to the cells so that the cells bind to each other rather than to the substrate. For example, in some embodiments, the microcavity is coated with an extremely low-binding material to make the microcavity non-adherent to the cells. The combination of non-adherent microcavities, spheroid-induced microcavity structures, and gravity can define a confinement volume that limits the growth of cells cultured within the microcavity, thereby forming spheroids with dimensions defined by the confinement volume. The spheroids expand to the size limit imposed by the microcavity structure. Because the microcavity has a uniform structure, cells growing within it can form cell aggregates or spheroids of similar size.

[0059] In some embodiments, the sidewalls, microcavities, the bottom of the microcavities, and / or other inner portions of the open wells are gas-permeable and liquid-impermeable. In some embodiments, the sidewalls, microcavities, the bottom of the microcavities, and / or other inner portions of the open wells are made of and / or coated with low-adhesion or non-adhesion materials. For example, in some embodiments, the inner surface of the open wells is treated with a polymer that inhibits cell adhesion to prevent cell adhesion. Non-limiting examples of such polymers include poly-HEMA, Pluronic®, or proprietary ULA treatment.

[0060] In some embodiments, the inner surface of the microcavity is non-adherent to cells. The microcavity may be formed from or coated with a non-adherent material to form a non-adherent well. Examples of non-adherent materials include perfluoropolymers, olefins, or similar polymers or mixtures thereof. Other examples include nonionic hydrogels such as agarose and polyacrylamide, polyethers such as polyethylene oxide, and polyols such as polyvinyl alcohol, or similar materials or combinations thereof. For example, a combination of a non-adherent well, well structure, and gravity can induce cells cultured in the well to self-organize into spheroids. Some spheroids can maintain the function of differentiated cells that exhibit a more in vivo-like response compared to cells grown in a monolayer.

[0061] In some embodiments, the microcavities are coated with materials having a low-binding treatment or extremely low-binding properties to make them non-adherent to cells. Examples of non-adherent materials include perfluoropolymers, olefins, or similar polymers or mixtures thereof. Other examples include nonionic hydrogels such as agarose and polyacrylamide, polyethers such as polyethylene oxide, and polyols such as polyvinyl alcohol, or similar materials or combinations thereof. For example, a combination of non-adherent microcavities, the structure of the microcavities (e.g., size and shape), and / or gravity can induce cells cultured within the microcavities to self-organize into spheroids. Some spheroids maintain the function of differentiated cells, exhibiting a more in vivo-like response compared to cells grown in a monolayer.

[0062] In some embodiments, the low-adhesion treatment or surface coating is a "Corning" Ultra Low Attachment (ULA) surface coating. This "Corning" ULA surface is hydrophilic, biologically inert, and non-degradable, thereby promoting highly reproducible spheroid formation and easy harvesting. The covalent bonding of the ultra-low adhesion surface reduces cell adhesion to the well surface. The ultra-low adhesion (ULA) surface enables uniform and reproducible 3D multicellular spheroid formation.

[0063] A wide variety of cell types can be cultured in the cell culture devices described herein. For example, any type of cell, including but not limited to immortalized cells, primary cultured cells, cancer cells, and stem cells (e.g., embryos or induced pluripotent cells), can be cultured in the open-well microcavity plate embodiments described herein. The cells may be mammalian cells, avian cells, fish cells, etc. The cells may be in any culture form, including dispersed (e.g., newly seeded), confluent, two-dimensional, three-dimensional, and spheroidal. Cultured cells may further be used in a variety of research, diagnostic, drug screening and testing, therapeutic, and industrial applications.

[0064] In some embodiments, the cells are mammalian cells (e.g., human, mouse, rat, rabbit, dog, cat, cow, pig, chicken, goat, horse, etc.). The cells may be of any type of tissue, including, but are not limited to, the kidney, fibroblasts, mammary gland, skin, brain, ovary, lung, bone, nerve, muscle, heart, colorectal, pancreas, immune system (e.g., B cells), blood, etc. The cells may be derived from any desired type of tissue or organ, including, but are not limited to, the adrenal gland, bladder, blood vessels, bone, bone marrow, brain, cartilage, cervix, cornea, endometrium, esophagus, digestive tract, immune system (e.g., T lymphocytes, B lymphocytes, leukocytes, macrophages, and dendritic cells), liver, lung, lymphatic vessels, muscle (e.g., cardiac muscle), nerve, ovary, pancreas (e.g., islet cells), pituitary gland, prostate, kidney, salivary gland, skin, tendon, testis, and thyroid gland. In some embodiments, the cells are somatic cells. In some embodiments, the cells are stem cells or progenitor cells (e.g., embryonic stem cells, induced pluripotent stem cells) in any desired differentiated state (e.g., pluripotent, multi-potent, fate-determined, immortalized, etc.). In some embodiments, the cells are disease cells or disease model cells. For example, in some embodiments, the spheroids include one or more types of cancer cells or cells that can be induced into a hyperproliferative state (e.g., transformed cells).

[0065] In some embodiments, the systems, devices, and methods described herein include one or more types of cells. In some embodiments, the cells are cryopreserved. In some embodiments, the cells are cultured in three dimensions. In some such embodiments, the systems, devices, and methods include one or more types of spheroids. In some embodiments, one or more types of cells are actively dividing. In some embodiments, the spheroids contain one type of cell. In some embodiments, the spheroids contain multiple types of cells. In some embodiments, when multiple spheroids are grown, each spheroid is of the same type, while in other embodiments, two or more different types of spheroids are grown. The cells growing within the spheroids may be native cells or mutated cells (e.g., cells containing one or more non-native genetic mutations).

[0066] When culturing cells using the cell culture devices described in these embodiments, any cell culture medium capable of supporting cell proliferation may be used. The cell culture medium may, for example, be sugars, salts, amino acids, serum (e.g., fetal bovine serum), antibodies, growth factors, differentiation factors, colorants, or other desired factors. Examples of cell culture media include Dulbecco's Modified Eagle Medium (DMEM), Ham F12 Nutrient Mixture, Minimum Essential Medium (MEM), RPMI medium, Iskov Modified Dulbecco's Medium (IMDM), and MesenCult™-XF medium (commercially available from STEMCELL Technologies Inc.).

[0067] In some embodiments, the system, device, and method include a culture medium (e.g., nutrients (e.g., proteins, peptides, amino acids), energy (e.g., hydrocarbons), essential metals and minerals (e.g., calcium, magnesium, iron, phosphates, sulfates), buffers (e.g., phosphates, acetates), pH change indicators (e.g., phenol red, bromocresol purple), and selectors (e.g., chemicals, antimicrobial agents). In some embodiments, one or more test compounds (e.g., drugs) are included in the system, device, and method.

[0068] Methods for culturing cells in embodiments of open-well microcavity plates described herein are also disclosed. In some embodiments, the method includes cell culture of cell aggregates or spheroids in the microcavity plate. Methods for culturing cells using microcavity plates described herein include the step of seeding cells into the microcavity plate. The step of seeding cells onto the microcavity plate may include the step of contacting the plate with a solution containing cells. The step of culturing cells on the microcavity plate may further include the step of contacting the microcavity plate with a cell culture medium. Generally, the step of contacting the microcavity plate with a cell culture medium includes seeding or placing the cells to be cultured onto the microcavity plate in an environment containing the medium in which the cells are to be cultured. The step of contacting the microcavity plate with a cell culture medium may include the step of supplying the cell culture medium onto the microcavity plate with a pipette.

[0069] In some embodiments, a method for culturing spheroids is provided, comprising the steps of introducing a culture medium into a cell culture device described herein, and adding spheroid-forming cells to the medium. In some embodiments, the method further comprises the step of replacing or exchanging the medium (e.g., daily). For example, the cell culture medium may be placed in the plate for a predetermined period of time. At least some of the cell culture medium may be removed after the predetermined period, and new cell culture medium may be added. The cell culture medium may be removed and exchanged according to any predetermined schedule. For example, at least some of the cell culture medium may be removed and exchanged hourly, or every 12 hours, or every 24 hours, or every 2 days, or every 3 days, or every 4 days, or every 5 days.

[0070] The cell culture apparatus described herein can be used in any suitable form to culture cells within the microcavities of the apparatus. For example, a method of culturing cells includes the step of introducing cells and cell culture medium into one or more of the microcavities of a cell culture apparatus such as those described herein. The step of culturing cells in one or more of the microcavities may include the step of forming spheroids within one or more microcavities. Spheroids cultured in one or more microcavities may be defined by a diameter of approximately, for example, 500 μm or less, 400 μm or less, 300 μm or less, 250 μm or less, 150 μm or less, or any range within the values ​​described above. The diameter of a single spheroid may differ from the average diameter of all spheroids grown in the microcavities by only approximately, for example, 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, or any range within the values ​​described above.

[0071] The open-well configuration facilitates manual handling and allows for the initial maintenance of a uniform culture environment. The fluid inlet area prevents spheroid disturbance due to the presence of pipette tips or turbulence from medium exchange. Cell culture medium can be replaced or exchanged as needed. A pipette can be used to introduce or remove medium from the microcavity plate. The tip of the pipette can be placed in the fluid inlet area to add cell culture medium.

[0072] Once the culture has formed the required characteristics, such as the number of cells or spheroids and their differentiation state, the cell culture medium in the plate may be removed. To remove the cell culture medium, the tip of a pipette can be inserted into the fluid inlet area. In some embodiments, some cell culture medium will remain in the individual microcavities along with the spheroids, so the cell culture medium may be mostly removed.

[0073] The methods of the present disclosure may further include a step of harvesting spheroids. Spheroids may be harvested in any suitable manner. For example, spheroids may be aspirated to remove them from a microcavity plate. In another example, gravity may be used to harvest spheroids from a microcavity plate. For example, in embodiments where the wells are non-adherent to cells, cells may be harvested by inverting the apparatus to move the cells out of the wells by gravity. Other non-limiting harvesting methods include scraping, vibration, and chemical means.

[0074] It will be recognized that various disclosed embodiments may include certain features, elements, or processes described in relation to a particular embodiment. It will also be recognized that, although certain features, elements, or processes are described in relation to a particular embodiment, they may be substituted or combined with alternative embodiments in various undescribed combinations or arrangements.

[0075] As used here, it should be understood that nouns refer to "at least one" object and should not be limited to "only one" unless otherwise specified. Therefore, for example, a reference to "opening" includes examples that have two or more such "openings," unless the context clearly indicates otherwise.

[0076] All scientific and technical terms used herein have their common meanings in the relevant technical field unless otherwise specified. The definitions provided herein are for the purpose of facilitating the understanding of certain terms used frequently and are not intended to limit the scope of this disclosure.

[0077] As used here, words like "to possess," "to include," and "to constitute" are used in an unrestricted sense, generally meaning "to include, but not limited to."

[0078] A range may be expressed here as "approximately" from one specific value and / or "approximately" to another specific value. When such a range is expressed, the example includes that specific value and / or the other specific value. Similarly, when a value is expressed as an approximation using the antecedent "approximately," it will be understood that the specific value forms another aspect. It will be further understood that each endpoint of a range is significant both in relation to the other endpoint and independently of the other endpoint.

[0079] All numerical values ​​expressed herein, unless otherwise specified, should be interpreted as including "approximately," whether or not they are explicitly stated as such. However, it will be further understood that each listed numerical value should be considered equally precise, regardless of whether or not it is expressed as "approximately." Therefore, both "dimensions less than 10 mm" and "dimensions approximately less than 10 mm" include embodiments of "dimensions approximately less than 10 mm" and "dimensions less than 10 mm."

[0080] Unless otherwise specified, none of the methods described herein are intended to be interpreted as requiring the steps to be performed in a specific order. Therefore, if a claim for a method does not actually enumerate the order in which the steps should be followed, or if it is not otherwise specifically stated in the claims or description that the steps should be limited to a particular order, no particular order is intended to be implied.

[0081] While various features, elements, or processes of a particular embodiment may be disclosed using the transitional phrase "includes," it should be understood that alternative embodiments are implied, including those that may be described using the transitional phrase "consist of" or "substantially consist of." Therefore, for example, alternative embodiments implied for a method including A+B+C include embodiments in which the method consists of A+B+C, and embodiments in which the method substantially consists of A+B+C.

[0082] While numerous embodiments of this disclosure have been described in detail, it should be understood that this disclosure is not limited to the disclosed embodiments and that various rearrangements, modifications, and substitutions are possible without departing from the disclosure as described and defined by the following claims.

[0083] Preferred embodiments of the present invention are described below in separate sections.

[0084] Embodiment 1 In cell culture devices, It is a frame, An open well located inside, and A fluid inlet area communicating with the aforementioned open well, A frame having The open well is provided, Upper opening, A base plate that constitutes the microcavity substrate and defines the main surface, and One or more side walls extending from the bottom plate to the upper opening, A cell culture device having the following features.

[0085] Embodiment 2 The cell culture device according to Embodiment 1, wherein the fluid inlet area includes the surface of one or more side walls.

[0086] Embodiment 3 The cell culture device according to Embodiment 2, wherein the surface of the side wall is inclined from the upper outer portion of the side wall to the bottom inner portion of the side wall along the length of the side wall.

[0087] Embodiment 4 The cell culture device according to Embodiment 3, wherein the upper outer portion is at the same level as the upper opening.

[0088] Embodiment 5 The cell culture device according to Embodiment 3, wherein the bottom inner portion is at the same level as the main surface and communicates with the main surface.

[0089] Embodiment 6 The cell culture device according to Embodiment 1, wherein the fluid inlet area has a notch located within one or more of the side walls.

[0090] Embodiment 7 The cell culture device according to Embodiment 6, wherein the notch includes a tetrahedral notch located in the center of the side wall.

[0091] Embodiment 8 The cell culture device according to Embodiment 7, wherein the edge of the tetrahedral notch is inclined from the upper outer portion of the side wall to the bottom inner portion of the side wall.

[0092] Embodiment 9 The cell culture device according to Embodiment 8, wherein the upper outer portion is at the same level as the upper opening.

[0093] Embodiment 10 The cell culture device according to Embodiment 8, wherein the bottom inner portion is at the same level as the main surface and communicates with the main surface.

[0094] Embodiment 11 The cell culture device according to Embodiment 1, wherein the fluid inlet area has a notch located at the corner of the open well, where the first side wall of the one or more side walls is joined at a right angle to the second side wall of the one or more side walls.

[0095] Embodiment 12 The cell culture device according to Embodiment 11, wherein the notch includes a tetrahedral notch located at the corner of the open well.

[0096] Embodiment 13 The cell culture device according to Embodiment 12, wherein the edge of the tetrahedral notch is inclined from the upper outer portion of the corner to the bottom inner portion of the corner.

[0097] Embodiment 14 The cell culture device according to Embodiment 13, wherein the upper outer portion of the corner is at the same level as the upper opening.

[0098] Embodiment 15 The cell culture device according to Embodiment 13, wherein the bottom inner portion of the corner is at the same level as the main surface and communicates with the main surface.

[0099] Embodiment 16 The cell culture device according to Embodiment 1, wherein the fluid inlet area includes a ledge positioned within one or more of the side walls.

[0100] Embodiment 17 The cell culture device according to embodiment 16, wherein the ledge is a grooved passage.

[0101] Embodiment 18 The cell culture device according to Embodiment 16, wherein the ledge is inclined from the top of the first end of the side wall to the bottom of the second end of the side wall.

[0102] Embodiment 19 The cell culture device according to Embodiment 18, wherein the bottom portion is at the same level as the main surface and communicates with the main surface.

[0103] Embodiment 20 The cell culture device according to Embodiment 18, wherein the upper part is at the same level as the upper opening.

[0104] Embodiment 21 The cell culture device according to Embodiment 1, wherein the fluid inlet area is the fluid outlet area.

[0105] Embodiment 22 The cell culture device according to Embodiment 1, wherein the cell culture device further includes a baffle.

[0106] Embodiment 23 The cell culture device according to embodiment 22, wherein the baffle is located in the open well between the main surface and the upper opening.

[0107] Embodiment 24 The cell culture device according to Embodiment 22, wherein the baffle comprises a plurality of baffle segments, each baffle segment extending from one end of the open well to the opposite end of the open well.

[0108] Embodiment 25 The cell culture device according to Embodiment 24, wherein at least one of the plurality of baffle segments is perpendicular to the other baffle segments.

[0109] Embodiment 26 The cell culture device according to Embodiment 24, wherein the first baffle segment is located in the open well adjacent to the fluid inlet area along the length of the side wall.

[0110] Embodiment 27 The cell culture device according to Embodiment 1, wherein the microcavity substrate comprises a plurality of microcavities.

[0111] Embodiment 28 The cell culture device according to Embodiment 27, wherein the plurality of microcavities are arranged in at least one row.

[0112] Embodiment 29 The cell culture device according to Embodiment 27, wherein the plurality of microcavities are arranged in a hexagonal close-packed pattern.

[0113] Embodiment 30 The cell culture device according to Embodiment 27, wherein each of the plurality of microcavities has an upper opening, a bottom, and a microcavity side wall surface extending from the upper opening to the bottom of the microcavity.

[0114] Embodiment 31 The cell culture device according to Embodiment 30, wherein the upper opening of the microcavity is coplanar with the main surface, and the bottom of the microcavity is located below the main surface.

[0115] Embodiment 32 A cell culture device according to Embodiment 30, wherein each microcavity has a rounded bottom.

[0116] Embodiment 33 The cell culture device according to Embodiment 30, wherein the width of the upper opening of each microcavity is 500 μm to 5 mm.

[0117] Embodiment 34 The cell culture device according to Embodiment 30, wherein the depth of each of the plurality of microcavities is 500 μm to 6 mm.

[0118] Embodiment 35 The cell culture device according to Embodiment 30, wherein each microcavity is non-adherent to cells.

[0119] Embodiment 36 The cell culture device according to Embodiment 35, wherein the internal surface of each microcavity is coated with an extremely low-adhesion material.

[0120] Embodiment 37 The cell culture device according to Embodiment 30, wherein each microcavity is designed so that cells cultured in the wells form spheroids.

[0121] Embodiment 38 The cell culture device according to Embodiment 1, wherein one or more side walls define a storage area on the microcavity substrate.

[0122] Embodiment 39 The cell culture device according to Embodiment 38, wherein one or more of the side walls have a height of 0.780 inches (approximately 20 mm).

[0123] Embodiment 40 The cell culture device according to Embodiment 1, wherein the inner surface of the open well is non-adherent to cells.

[0124] Embodiment 41 The cell culture device according to Embodiment 40, wherein the inner surface of the open well includes a non-adhesive surface coating made from a perfluoropolymer, olefin, agarose, nonionic hydrogel, polyether, polyol, polymer that inhibits cell adhesion, or a combination thereof.

[0125] Embodiment 42 The cell culture device according to Embodiment 41, wherein the non-adhesive surface coating includes an extremely low adhesion (ULA) surface coating.

[0126] Embodiment 43 The cell culture device according to Embodiment 1, wherein the frame, one or more side walls, or a combination thereof, are formed from polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polymethylpentene, polycarbonate, polymethyl methacrylate, styrene-ethylene-butadiene-styrene, silicone rubber or copolymer, ethylene vinyl acetate, polysulfone, polytetrafluoroethylene, poly(styrene-butadiene-styrene), or a combination thereof.

[0127] Embodiment 44 The cell culture device according to Embodiment 1, wherein the microcavity substrate is formed from polydimethylsiloxane (PDMS), polymethylpentene, (poly)4-methylpentene (PMP), polyethylene (PE), polystyrene (PS), polypropylene, polyethylene terephthalate, polycarbonate, polymethyl methacrylate, styrene-ethylene-butadiene-styrene, silicone rubber or copolymer, ethylene vinyl acetate, polysulfone, polytetrafluoroethylene, poly(styrene-butadiene-styrene), or a combination thereof.

[0128] Embodiment 45 The cell culture device according to Embodiment 1, wherein the cell culture device is a storage open-well type microcavity plate. [Explanation of Symbols]

[0129] 100, 200, 300, 400 microcavity plates 102, 302 frames 105, 205, 305, 405 Fluid inlet area 120, 121, 122, 123, 220, 320, 321, 420, 421 One or more side walls 124, 224, 334 Upper outer part 126, 336 Bottom inner part 150, 350 open wells 155 Upper opening 160, 260, 360, 460, 900, 1000, 1100 microcavity substrates 161, 261, 361, 461 Main surface 162 Bottom plate 170 Skirt 190, 290, 390, 490 baffles 191, 193, 195, 291, 293, 295, 391, 393, 395, 491, 493, 495 baffle segments 241, 341 Edges 407 Grooved walkway 500 Spheroids 910, 1001, 1101 Microcavities

Claims

[Claim 1] In cell culture devices, It is a frame, An open well located inside, and A fluid inlet area communicating with the open well and having a tetrahedral notch, A frame having The open well is provided, Upper opening, A base plate that constitutes the microcavity substrate and defines the main surface, and One or more side walls extending from the bottom plate to the upper opening, A cell culture device having the following features.

Images