Microcavity plate

Abstract

The microcavity plate comprises a microcavity substrate and a base (303). The base comprises a lower grid comprising a plurality of grid segments arranged in a regular matrix to form a plurality of openings, and an open well (307) comprising a plurality of sidewalls (309) extending vertically from the periphery of the lower grid. The microcavity substrate comprises a plurality of microcavities (315) arranged in a regular matrix aligned with the plurality of openings in the lower grid, each microcavity having a cavity disposed within an opening in the lower grid. The microcavity plate can further comprise an upper grid (405) configured to be disposed on the lower grid that defines the regular matrix of the base.

Description

Cross - reference to related applications

[0001] This application claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 63 / 107,663, filed October 30, 2020, and all disclosures of this provisional application are relied upon and incorporated herein by reference in their entirety.

Technical Field

[0002] The present disclosure generally relates to micro - cavity plates. Specifically, the present disclosure relates to micro - cavity plates used for cell culture and high - throughput screening.

Background Art

[0003] For many diseases, the first step in the process of discovering a treatment is the discovery and development of drugs. In this step, many molecular compounds that are candidates for drug therapy are tested. Due to the large number of tests, this test is often carried out using high - throughput screening (HTS). HTS is a method that uses automated equipment to rapidly test the biological activity of countless samples. In HTS, microplates are often used, and a typical configuration of an automatable microplate is a 1536 - well plate.

[0004] Furthermore, to more accurately reproduce the environment that cells experience in vivo, HTS can be performed using a three-dimensional (3D) cell culture model (spheroid). Spheroids (3D cell cultures) generally have a diameter in the range of approximately 100 to 300 micrometers. Conventional techniques allow for dispensing spheroids into 96-well or 384-well plates. However, because spheroids are large, there was a risk that they could not be dispensed into the wells of a 1536-well plate due to the well shape of the 1536-well plate. Therefore, conventional 1536-well cell culture devices that generate 3D cell cultures (spheroids) were generally not suitable for automating operations in the HTS process. Moreover, conventional equipment did not have the capability to dispense large structures such as spheroids cultured in bulk spheroid production vessels into 1536-well plates for HTS. [Overview of the project]

[0005] Embodiments of this disclosure provide a microcavity plate for bulk spheroid preparation that can also be used in the HTS process. The microcavity plate comprises 1536 shallow cavities, each with a diameter of 1500 μm. These cavities are arranged in a regular matrix similar to that of a typical 1536-well plate. In several embodiments, by adding a grid to the top surface of the microcavities, it becomes possible to treat each microcavity individually and perform HTS. Therefore, embodiments of this disclosure enable individual treatment of spheroids that have been cultured together from the start of the culture process after the grid has been placed, thereby solving problems with existing conventional equipment.

[0006] In one embodiment, the microcavity plate comprises a base and a microcavity substrate. The base comprises a lower grid having a plurality of grid segments arranged in a regular matrix to form a plurality of openings, and an open well having a plurality of side walls extending vertically from the outer periphery of the lower grid. The microcavity substrate comprises a plurality of microcavities arranged in a regular matrix aligned with the plurality of openings of the lower grid, each microcavity having a cavity positioned within the openings of the lower grid.

[0007] In some embodiments, the microcavity plate further comprises an upper grid. In some embodiments, the upper grid comprises a plurality of well openings in a regular matrix that mirrors the regular matrix of the lower grid. In some embodiments, the upper grid is configured to be positioned on a lower grid that demarcates a regular matrix at the base within the open wells. In some embodiments, the plurality of well openings are aligned with a plurality of microcavities of the microcavity substrate. In some embodiments, a microcavity well is defined by a plurality of side walls composed of grid segments defining each microcavity opening and an individual microcavity positioned at the center of the microcavity opening.

[0008] In some embodiments, the microcavity plate further comprises a gasket material. In some embodiments, the gasket material is integral with the bottom surface of the upper grid, and when the upper grid is inserted into the open well, the gasket material is positioned between the bottom surface of the upper grid and the top surface of the microcavity substrate.

[0009] In some embodiments, the multiple microcavities include 1536 individual microcavities.

[0010] In some embodiments, each of the multiple microcavities has a top surface and a rounded bottom. In some embodiments, the diameter of each microcavity at its top surface is approximately 1500 μm.

[0011] In some embodiments, the inner surface of the cavity is coated with a non-adherent film to cells. In some embodiments, the film includes an ultra-low adhesion (ULA) surface coating.

[0012] In some embodiments, the upper grid comprises a plurality of projections extending from the outer periphery of the upper grid. In some embodiments, a plurality of the projections are configured to align with and interlock with a plurality of through holes provided in the side wall of the open well.

[0013] In some embodiments, the microcavity substrate is formed from a polymer selected from polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polymethylpentene, polycarbonate, polymethyl methacrylate, styrene-butadiene copolymer, styrene-ethylene-butylene-styrene, other similar polymers, or combinations thereof. In some embodiments, the microcavity substrate is formed from polystyrene.

[0014] In some embodiments, the base is formed from a polymer including polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polymethylpentene, polycarbonate, polymethyl methacrylate, styrene-butadiene copolymer, styrene-ethylene-butylene-styrene, other similar polymers, or combinations thereof. In some embodiments, the base is formed from polystyrene.

[0015] In some embodiments, the upper grid is formed from an elastomer material selected from natural rubber, styrene-butadiene block copolymer, styrene-ethylene-butylene-styrene polymer, polyisoprene, polybutadiene, ethylene propylene rubber, ethylene propylene diene rubber, silicone elastomer, fluoroelastomer, polyurethane elastomer, nitrile rubber, or a combination thereof.

[0016] In some embodiments, the gasket material is formed from an elastomer. In some embodiments, the elastomer contains silicone. In some embodiments, the gasket material is a pressure-sensitive adhesive.

[0017] In some embodiments, microcavity plates are used for spheroid cell culture. In some embodiments, microcavity plates are used for high-throughput screening.

[0018] In one embodiment, a method for performing high-throughput screening includes the step of seeding cells into a microcavity plate described herein. The method further includes the steps of culturing the cells to form spheroids in a plurality of microcavities, attaching an upper grid to the top of the plurality of microcavities in the microcavity plate to form microcavity wells, and performing high-throughput screening of the cultured spheroids by treating each microcavity well individually. In some embodiments, the step of culturing the cells includes the step of bringing the cells in the microcavity plate into contact with a cell culture medium. [Brief explanation of the drawing]

[0019] [Figure 1] Image showing a standard 1536-well plate. [Figure 2] Image showing a configuration in which multiple microcavities are arranged in a hexagonal close-packed arrangement. [Figure 3]Top view showing a 1536 - micro - cavity plate according to an embodiment of the present disclosure [Figure 4] Top view showing a grid for a 1536 - micro - cavity plate according to an embodiment of the present disclosure [Figure 5] Top view showing a 1536 - micro - cavity plate with a grid according to an embodiment of the present disclosure [Figure 6] Top view showing a 1536 - micro - cavity plate with a grid according to an embodiment of the present disclosure [Figure 7] Side - sectional view showing a 1536 - micro - cavity plate according to an embodiment of the present disclosure [Figure 8] Side - sectional view showing a 1536 - micro - cavity plate with a grid according to an embodiment of the present disclosure [Figure 9] Side - sectional view showing a 1536 - micro - cavity plate with a grid and a gasket according to an embodiment of the present disclosure [Figure 10] Side - sectional view showing a 1536 - micro - cavity plate with a grid and a gasket according to an embodiment of the present disclosure

Mode for Carrying Out the Invention

[0020] To more accurately reproduce the environment that cells experience in vivo, HTS can be performed using three-dimensional cell culture models. Recent studies have revealed that cellular responses in three-dimensional cultures, such as three-dimensional spheroids and three-dimensional organoids (hereinafter referred to as spheroids), are closer to in vivo behavior than cellular responses in two-dimensional (2D) cultures where cells are cultured in a single layer. It is thought that changes in cellular responses occur in three-dimensional cultures due to the expansion of dimensions. This is because, as a result of the three-dimensional expansion, the spatial configuration of receptors on the cell surface that are responsible for interactions with surrounding cells is affected, imposing physical constraints on the cell. This, in turn, affects extracellular to intracellular signal transduction, and ultimately influences gene expression and cell behavior. However, conventional culture devices for generating three-dimensional cell cultures (spheroids) were not optimal for automating operations in the HTS process.

[0021] In HTS, microplates are often used, and a typical configuration of a microplate capable of automated operation is the 1536-well plate. Figure 1 is an image showing a standard 1536-well plate 10. The 1536-well plate shown in Figure 1 has multiple wells 15 arranged in a matrix, with individual wells 13 spaced equally apart. The working surface area of ​​the 1536-well plate is approximately 4.5 inches (approximately 114.3 mm) x approximately 3 inches (approximately 76.2 mm), and in this microplate, the wells are arranged in a matrix at equal intervals across this entire working surface area. The wells of the 1536-well plate have a diameter of approximately 1500 μm and are very deep (approximately 6000 μm), so each well must be handled individually, making manual operation difficult.

[0022] In addition, while microplate wells are in the millimeter (mm) or centimeter (cm) size range, conventional culture devices for generating three-dimensional cell cultures (spheroids) have wells in the micrometer size range. Culture devices with micrometer-sized wells are referred to by various names such as microcavities and microspaces. A microcavity typically has an internal cavity with a rounded non-cell-adhesive bottom, and supports three-dimensional cell culture by enabling spheroid formation within each microcavity through self-organization (adhesion to each other) of cells seeded within the microcavity. A typical microcavity is shallow (about 500 to about 3000 μm), and since all spheroids within all cavities can be covered simultaneously with cell culture medium, manual operation is easy.

[0023] A microcavity container is called a bulk spheroid production container, and enables culturing of many spheroids at once due to the micrometer-sized shape of the wells and the hexagonal close-packed ("honeycomb") well arrangement. Figure 2 is an image showing a configuration in which a plurality of microcavities 20 are arranged in a hexagonal close-packed arrangement 25. With such a close-packed arrangement, it is possible to arrange approximately 12,588 wells with a diameter of 500 μm on a working surface area of a typical microplate of 4.5 inches (about 114.3 mm) × 3 inches (about 76.2 mm). Also, when the diameter of the microcavity is increased up to 1500 μm (the diameter of the wells of a 1536-well plate), by arranging the wells in a hexagonal close-packed arrangement, approximately 2,845 wells can be arranged on the working surface area of a typical 4.5-inch (about 114.3 mm) × 3-inch (about 76.2 mm) microplate. However, while bulk spheroid production is possible with wells in a hexagonal close-packed arrangement, it could not be used with standard equipment used in HTS. This is because conventional bulk spheroid production containers did not have a configuration for automatically operating wells arranged at equal intervals in a matrix.

[0024] Embodiments of this disclosure provide a microcavity plate for bulk spheroid preparation that can also be used in the HTS process. The microcavity plate has 1536 shallow cavities, each with a diameter of 1500 μm. These cavities are arranged in a regular matrix similar to that of a typical 1536-well plate. Because the microcavities are arranged in a regular matrix, an upper grid can be added to the top surface of the microcavities. By adding the upper grid, each microcavity becomes a well, allowing each to be treated individually, thus enabling HTS. Therefore, embodiments of this disclosure enable the cultivation of multiple spheroids together from the start of the culture process to create a homogeneous culture environment, and after the grid is placed, it is possible to treat each spheroid individually.

[0025] The microcavity plate according to the embodiment of this disclosure provides a homogeneous culture environment. Before the upper grid is placed on the microcavity plate to form individual wells, the same treatment can be performed on all spheroids cultured in the microcavity plate having 1536 wells at the same time, thus providing a homogeneous culture environment. In contrast, with a general plate having individual wells, dispensing the same amount into each well is difficult even with automated equipment, resulting in a low homogeneity of the culture environment.

[0026] The microcavity plate according to the embodiment described herein can be converted into a plate having individual wells by adding an upper grid. The upper grid can be positioned above the multiple microcavities in the plate. The configuration of providing multiple individual wells in the microcavity plate allows for individual handling of spheroids in each well by adding a grid in a later process, thus enabling the user to perform HTS.

[0027] The microcavity plates of the embodiments described herein do not require an automated dispenser for handling spheroids (transferring spheroids from a bulk spheroid preparation container to a 1536-well plate).

[0028] Figure 3 is an overhead view showing a 1536 microcavity plate without the upper grid. The microcavity plate 300 has a base 303. An open well 307 is positioned on the base 303. This rectangular open well 307 is defined by four side walls 309 extending vertically from the base 303. Multiple microcavities 315 are positioned within the open well 307. Each microcavity 310 is a shallow cavity with a diameter of approximately 1500 μm. The multiple microcavities 315 are regularly arranged in a matrix of multiple rows 325 and multiple columns 335. Each row 320 is parallel to the other columns in the multiple rows 325. Similarly, each column 330 is parallel to the other columns in the multiple columns 335. In the 1536 microcavity plate, each row 320 consists of 48 microcavities arranged equally apart in a first direction, and each column 330 consists of 32 microcavities arranged equally apart in a second direction. The second direction is perpendicular to the first direction, thereby forming a grid pattern from multiple rows 325 and multiple columns 335.

[0029] Figure 4 is an overhead view showing an upper grid used in a 1536 microcavity plate. The upper grid 405 has a rectangular shape that can be positioned inside the four side walls of the open wells of the microcavity plate. The upper grid 405 comprises a plurality of grid segments 411 in a first direction and a plurality of grid segments 413 in a second direction. The grid segments 411 in the first direction are positioned perpendicular to the grid segments 413 in the second direction, forming a grid pattern. These grid segments form a grid of openings arranged regularly in a grid pattern of a plurality of rows 425 and a plurality of columns 435. Each row 420 consists of 48 openings arranged at equal intervals, and each column 430 consists of 32 openings arranged at equal intervals. The size of the upper grid 405 is set so that each individual microcavity in the microcavity plate is individually surrounded by its respective opening 417. Once the grid is positioned in place on the microcavity plate, the upper grid forms the sidewalls of each microcavity well, creating a plurality of individual microcavity wells.

[0030] Figure 5 is an overhead view showing the 1536 microcavity plate with the upper grid positioned in place. The 1536 microcavity plate 500 with the upper grid device attached, as shown in Figure 5, has 1536 individual microcavity wells 375. The individual microcavity wells 375 are regularly arranged in a matrix of multiple columns 535 and multiple rows 525. Each microcavity well column 530 consists of 32 microcavity wells. Each microcavity well row 520 consists of 48 microcavity wells.

[0031] Figure 6 is an overhead view showing one embodiment of the 1536 microcavity plate 600 with the upper grid positioned in place. The dimensions of the embodiment of the 1536 microcavity plate 600 shown in Figure 6 are shown as examples of dimensions that can be used for the 1536 microcavity plate described herein, and are not limited to these. The footprint of the microcavity plate according to the embodiments described herein can be the footprint of the standard dimensions of a conventional 1536 well plate. Examples of such standard footprints include the standard footprint dimensions of a 1536 well plate specified by the American National Standards Institute (ANSI) and the Society for Biomolecular Sciences (SBS). For example, in some embodiments, the length of the microcavity plate is approximately 5.0299 inches (approximately 127.759 mm). In some embodiments, the width of the microcavity plate is approximately 3.3654 inches (approximately 85.481 mm). In some embodiments, the working area of ​​the microcavity plate is defined by an open well area having a length of approximately 4.252 inches (approximately 108.0 mm) and a width of approximately 2.8347 inches (approximately 72.001 mm). In several embodiments, each microcavity well has a length of approximately 0.0886 inches (approximately 2250.4 μm) and a width of approximately 0.0886 inches (approximately 2250.4 μm), with individual microcavities located at the center of each microcavity well. In several embodiments, each microcavity has a diameter of approximately 1500 μm (0.05906 inches). While the height of a standard 96-well or 384-well plate is 0.560 inches (approximately 14.22 mm), in several embodiments, the height of the microcavity plate is approximately 0.780 inches (approximately 19.81 mm). The exemplary dimensions shown herein are not intended to be limiting, and their dimensional tolerances are approximately ±0.010 inches (approximately ±254 μm).

[0032] Figure 7 is a side cross-sectional view showing a portion of the 1536 microcavity plate. In the illustrated portion of the microcavity plate 700, a molded bottom component (base) 10 is shown. The base 10 has an open well defined by a side wall 13 and a lower grid 17. The base 10 may have a flat bottom and a flange (skirt) 12 provided on the outer circumference of the bottom of the base 10. A microcavity substrate 20 having a recess is placed inside the base 10, and individual microcavities 23 are individually fitted into the openings of the lower grid 17. The lower grid 17 may comprise a plurality of grid segments arranged in a regular matrix, each grid segment may have a flat top and a flat bottom. The base 10 is further defined by an open well having a side wall 13 that extends vertically around the outer circumference of the lower grid and forms an outer periphery surrounding the lower grid. The microcavity substrate 20 is placed in the open well so that the openings of the lower grid 17, which is arranged in a regular matrix configuration, are aligned with the openings of the lower grid 23, which is arranged in a regular matrix configuration. In some embodiments, the height of the lower grid can be about 0.060 inches (about 1524.0 μm). In some embodiments, the height of the lower grid can be about 0.063 inches (about 1600.2 μm), and each opening of the lower grid is about 0.0886 inches (about 2250.4 μm) in length and about 0.0886 inches (about 2250.4 μm) in width. The microcavity substrate 20 comprises a plurality of microcavities 23, each having a curved (rounded) bottom 27, which defines a recessed microcavity. Each microcavity has a cavity with a circular top opening and a rounded bottom. Each microcavity has a diameter of approximately 1500 μm and a depth of approximately 1600 μm. The microcavity substrate 20 is placed into the open well so that each microcavity of the microcavity substrate is centered on the opening of the lower grid. The bottom of the microcavity can be positioned above the bottom surface of the microcavity plate.

[0033] Microcavity substrates can be formed from film material. For example, a microcavity substrate can be formed from a flat film material having a thickness of 0.003 to 0.015 inches (approximately 76.2 to 381.0 μm), but is not limited to this. The film material can be formed from any suitable material. Examples of such materials include, but are not limited to, polystyrene, polymethylpentene, polyethylene, polypropylene, or laminates. The thickness at the top of the rounded bottom of the microcavity substrate can be in the range of approximately 35 micrometers to approximately 75 micrometers. The thickness of other parts of the microcavity substrate may vary depending on the location.

[0034] Figure 8 is a side cross-sectional view showing a portion of the 1536 microcavity plate. The illustrated portion of the microcavity plate 800 shows the upper grid 30 positioned on the upper part 25 of the microcavity substrate 20 within the open well of the base 10. By positioning the upper grid 30 on the microcavity substrate 20 and adding it to the molded bottom component of the plate, the segments of the upper grid form the side walls 55 of the microcavity wells 50, thereby defining individual microcavity wells 50. The positioning of the upper grid 30 within the open well can be carried out such that the lower part (bottom) 35 of the upper grid 30 is aligned with the lower grid of the microcavity plate and positioned on the upper part 25 of the microcavity substrate 20, while the upper part 33 of the upper grid 30 extends to the upper part 11 of the base 10 or the upper part of the open well side wall. The upper grid 30 has a regular matrix formed by a plurality of grid segments 37 in a first direction and a plurality of grid segments 39 in a vertical direction. In some embodiments, the upper grid has a height of approximately 0.717 inches (approximately 18.212 mm). Multiple grid segments of the upper grid 30 define multiple openings, each approximately 0.0886 inches (approximately 2250.4 μm) in length and approximately 0.0886 inches (approximately 2250.4 μm) in width, which mirror the lattice configuration of the lower grid 17. Optionally, the base may also include a flange (skirt) 12 along the outer periphery (outer edge) of the microcavity plate. This configuration can contribute to the stability of the plate.

[0035] Figure 9 is a side cross-sectional view showing a portion of a 1536 microcavity plate equipped with a gasket sheet according to an embodiment of the present invention. As shown in Figure 9, a gasket material (gasket sheet) 60 can be used to seal the space between the wells 50. The gasket sheet 60 is positioned between the upper part 25 of the microcavity substrate 20 and the lower part 35 of the upper grid 30. In several embodiments, the gasket material (gasket sheet) is formed from an elastomer. In some examples, the gasket sheet is formed from silicone. The gasket sheet can be used to seal the portion where the upper grid contacts the upper surface of the microcavity substrate. Such sealing can be achieved, for example, by snapping the plates together and applying compression, or by using an adhesive-based gasket sheet made from a material such as a pressure-sensitive adhesive. In some embodiments, a snap-fit ​​mechanism is provided on the outer circumference of both plates. In some embodiments, a snap-fit ​​mechanism is provided inside the plates. This snap-fit ​​mechanism is provided in the form of pillars between four adjacent wells, compressing and fitting these four wells together.

[0036] Figure 10 is a side cross-sectional view showing a portion of the 1536 microcavity plate with the upper grid 30 sealed by a gasket 60. The gasket material 60 is positioned between the lower part 35 of the upper grid 30 and the upper part 25 of the microcavity substrate 20. The upper grid 30 has projections 31 on its outer circumference. The projections 31 are configured to snap into place by engaging with through holes 61 provided in the side wall 13 of the open well (i.e., the outer periphery wall of the plate). When compression is applied (pressed into place), the projections of the upper grid engage with the through holes in the outer periphery wall of the plate, sealing the upper grid to a portion of the plate. As a result, multiple microcavities are transformed into multiple microcavity wells.

[0037] In some embodiments, the upper grid can be integrally molded as a single piece. In some embodiments, the bottom component, comprising a base, a lower grid, and a microcavity substrate, can be integrally molded as a single piece. The gasket material can be overmolded onto the upper grid. In some embodiments, the bottom component can be formed by injection molding. The gasket material can be any suitable material, such as an elastomer. In some embodiments, the elastomer can be silicone. In some embodiments, the gasket material is a pressure-sensitive adhesive.

[0038] The microcavity wells can have any suitable non-bonding coating. For example, such a coating can be a non-adherent surface coating to cells. In some embodiments, such a non-cell-adherent surface coating is Corning's Ultra Low Attachment (ULA) surface coating. Corning's Ultra Low Attachment surface is hydrophilic, non-biologically active, and non-degradable, contributing to the formation of highly reproducible spheroids and their easy harvesting. The covalent bonding of the Ultra Low Attachment surface reduces cell adhesion to the well surface. The Ultra Low Attachment (ULA) surface enables the formation of homogeneous and highly reproducible three-dimensional multicellular spheroids. This 1536 microcavity well configuration enables three-dimensional cell culture and analysis with high production efficiency.

[0039] The microcavity plates according to the embodiments described herein can be formed from any suitable material. In some examples, the formation of the microcavity plate can be carried out in multiple steps. For example, one step may be the construction of the bottom component of the 1536 microcavity plate. The bottom component is a component comprising a base and a lower grid portion of the plate. Materials used for such construction include plastic polymers, copolymers, or polymer blends. Examples, but not limited to, include polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polymethylpentene, polycarbonate, polymethyl methacrylate, styrene-butadiene copolymer, styrene-ethylene-butylene-styrene, other similar polymers, or combinations thereof. Furthermore, the lower grid or base of the microplate can be formed using any suitable construction method. Examples, but not limited to, include injection molding, thermoforming, or methods suitable for molding plastic parts such as 3D printing.

[0040] Furthermore, the microcavity substrate can be formed simultaneously with the lower grid section. The microcavity substrate can be formed using the same or similar materials and methods as those used to fabricate the other parts of the microcavity plate. In some embodiments, the microcavity substrate can be molded (formed) separately from the other parts of the plate and then joined. Such joining can be performed by any plastic joining method, such as thermal joining or ultrasonic welding. Examples of materials used to construct the microcavity substrate include plastic polymers, copolymers, or polymer blends. Examples, but not limited to, include polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polymethylpentene, polycarbonate, polymethyl methacrylate, styrene-butadiene copolymer, styrene-ethylene-butylene-styrene polymer, other similar polymers, or combinations thereof. The microcavity substrate can also be formed using any suitable construction method. Examples of such construction methods include, but are not limited to, injection molding, thermoforming, or methods suitable for molding plastic parts, such as 3D printing.

[0041] The upper grid can be formed using the same material as the base or lower grid and the microcavity substrate. In some embodiments, the upper grid can also be formed using a more elastic elastomer material, such as natural rubber, styrene-butadiene block copolymer, styrene-ethylene-butylene-styrene polymer, polyisoprene, polybutadiene, ethylene propylene rubber, ethylene propylene diene rubber, silicone elastomer, fluoroelastomer, polyurethane elastomer, or nitrile rubber. The upper grid of the microplate can also be formed using any suitable construction method. Examples of such construction methods include, but are not limited to, methods suitable for molding plastic parts, such as injection molding, thermoforming, or 3D printing.

[0042] In some embodiments where the top grid is made from a non-elastomer material, the manufacturing process of the top grid includes an additional step of adding an elastomer to the portion of the grid that is in direct contact with the substrate. This elastomer facilitates sealing between the substrate and the top grid, maintaining the integrity of the individual microcavity wells after the top grid is installed. In such embodiments, the top grid is supplied packaged separately from the microcavity plate. This is because the top grid needs to be kept sterile while cell culture is being performed using the rest of the plate.

[0043] Embodiments of this disclosure also disclose methods for culturing or harvesting cells on a microcavity plate as described herein. In some embodiments, the method includes cell culture of cell aggregates (spheroids) in a microcavity plate.

[0044] Embodiments of this disclosure further include a method of using the microcavity plate described herein. When using the 1536 microcavity plate, the user must remove the top grid from the microcavity plate before seeding cells into the microcavity plate. This facilitates manual operation and allows for the maintenance of a homogeneous culture environment in the initial stages. Once the necessary characteristics (cell number, spheroid number, differentiation state, etc.) are generated through culture, the cell culture medium in the plate is drained. Since some cell culture medium naturally remains in the individual microcavities containing spheroids, the draining process at this stage results in almost complete removal of the cell culture medium. At this point, the top grid can be inserted, and culture medium and reagents can be added to perform HTS.

[0045] In one embodiment, a method for performing high-throughput screening includes the steps of: seeding cells into a microcavity plate; culturing the cells to form spheroids in a plurality of microcavities; attaching an upper grid to the top of the plurality of microcavities in the microcavity plate to form microcavity wells; and performing high-throughput screening of the cultured spheroids by handling each microcavity well individually. The step of culturing cells may include bringing the cells in the microcavity plate into contact with a cell culture medium.

[0046] Microcavity plates can be used to culture any type of cell. These cells include, but are not limited to, immortalized cells, primary cultured cells, cancer cells, and stem cells (e.g., embryonic cells or induced pluripotent cells). The cells may be mammalian cells, avian cells, fish cells, etc. Furthermore, the cells can be any tissue cells. Examples include, but are not limited to, cells from the kidney, fibroblasts, breast, skin, brain, ovaries, lungs, bone, nerves, muscles, heart, colorectal, pancreas, immune cells (e.g., B cells), and blood. The cells can be cultured in any form, such as dispersed (e.g., newly seeded cells), confluent, 2D culture, 3D culture, or spheroids.

[0047] The step of culturing cells on a microcavity plate may include the step of seeding cells on the microcavity plate. The step of seeding cells on a microcavity plate may include the step of contacting the microcavity plate with a solution containing the cells. The step of culturing cells on a 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 the step of seeding (placing) the cells to be cultured on the microcavity plate into an environment having a culture medium for cell culture. The step of contacting the microcavity plate with a cell culture medium may include the step of pipetting the cell culture medium into the microcavity plate. In some embodiments, the cell culture medium can be kept in the microcavity plate for a predetermined period of time, after which at least a portion of the cell culture medium can be drained and fresh cell culture medium can be added. Drainage or replacement of the cell culture medium can be carried out according to any predetermined schedule. For example, at least a portion of the cell culture medium can be drained and replaced every hour, every 12 hours, every 24 hours, every 2 days, every 3 days, every 4 days, or every 5 days.

[0048] Any cell culture medium capable of supporting cell proliferation can be used. Examples of cell culture media include, but are not limited to, desired factors such as sugars, salts, amino acids, serum (e.g., fetal bovine serum), antibiotics, growth factors, differentiation factors, or colorants. Exemplary cell culture media include Dulbecco's Modified Eagle Medium (DMEM), Ham's F12 Nutrient Mixture, Minimum Essential Media (MEM), RPMI medium, Iscove's Modified Dulbecco's Medium (IMDM), and MesenCult-XF medium (commercially available from STEMCELL Technologies Inc.).

[0049] While specific features, elements, or steps have been described above in relation to specific embodiments, it will be understood that these can be incorporated into various embodiments of this disclosure. It will also be understood that specific features, elements, or steps described in relation to one particular embodiment can be interchanged or combined with those of other embodiments in various combinations or sequences not illustrated.

[0050] In this specification, “the,” “a,” or “an” means “at least one,” and should not be limited to meaning “only one” unless otherwise explicitly stated. Therefore, for example, the expression “opening” introduced by the article “a” also includes examples where there are two or more “openings,” unless it is clearly otherwise in the context.

[0051] All scientific and technical terms used herein have their meanings as commonly used in the art, unless otherwise specified. The definitions provided herein are for the purpose of facilitating the understanding of certain terms that are used repeatedly herein and are not intended to limit the scope of this disclosure.

[0052] In this specification, "have," "including," and "comprise" (such as have, having, include, including, comprise, comprising) are used in an open-ended sense, generally meaning "including, but not limited to."

[0053] In this specification, ranges may be expressed as "about" greater than or equal to a certain value, "about" a certain value to "about" another specific value, or "about" less than or equal to the other specific value. When ranges are expressed in this way, other embodiments exist that include the range from the certain value to the other specific value. Similarly, when a value is expressed as an approximation by placing "about" before it, it will be understood that other embodiments consisting of that specific value itself also exist. Furthermore, it will be understood that the meanings of the two endpoints of each range are both correlated and independent of each other.

[0054] Unless otherwise specified, all numerical values ​​described herein, whether accompanied by the word "approximately" or not, should be interpreted as including "approximately." However, it should also be understood that all numerical values ​​described herein, whether expressed as values ​​derived with "approximately" or not, have been carefully considered. Therefore, both "dimensions less than 10 mm" and "dimensions less than approximately 10 mm" encompass both embodiments of "dimensions less than approximately 10 mm" and embodiments of "dimensions less than 10 mm."

[0055] Unless otherwise specified, no method described herein is intended to be construed as requiring each step (process) to be performed in a specific order. Therefore, unless the order of the steps is actually described in the method claim, or unless there is other clear indication in the claims or detailed description of the invention that each step is limited to a specific order, it is not intended that any specific order of the steps be inferred.

[0056] While various features, elements, or steps of a particular embodiment may be disclosed using the transitional phrases "comprising," "comprising," or "having," it should be understood that this also implies alternative embodiments, such as those that could be described using the transitional phrases "consisting" or "consisting essentially of." For example, alternative embodiments implied by a method comprising A+B+C include embodiments of a method consisting of A+B+C and embodiments of a method consisting essentially of A+B+C.

[0057] While several embodiments of this disclosure have been described in detail above, it should be understood that this disclosure is not limited to the embodiments described herein, and that a wide variety of reconfigurations, modifications, and substitutions are possible, as long as they do not deviate from the disclosure described and defined by the appended claims.

[0058] Preferred embodiments of the present invention are described below in separate sections.

[0059] Embodiment 1 It is a microcavity plate, A lower grid comprising multiple grid segments that form multiple openings by being arranged in a regular matrix, An open well comprising a plurality of side walls extending vertically from the outer periphery of the lower grid, A base equipped with, A microcavity substrate comprising a plurality of microcavities, each having a cavity positioned within the openings of the lower grid, arranged in a regular matrix with respect to the plurality of openings of the lower grid, A microcavity plate equipped with [a specific feature / feature].

[0060] Embodiment 2 A microcavity plate according to Embodiment 1, further comprising an upper grid.

[0061] Embodiment 3 The microcavity plate according to Embodiment 2, wherein the upper grid comprises a plurality of well openings in a regular matrix that mirrors the regular matrix of the lower grid.

[0062] Embodiment 4 The microcavity plate according to Embodiment 3, wherein the upper grid is positioned on the lower grid that demarcates the regular matrix of the base within the open well.

[0063] Embodiment 5 The microcavity plate according to Embodiment 4, wherein the plurality of well openings are aligned with the plurality of microcavities of the microcavity substrate.

[0064] Embodiment 6 The microcavity plate according to Embodiment 5, wherein a microcavity well is defined by a plurality of side walls composed of grid segments defining each microcavity opening, and an individual microcavity positioned at the center of the microcavity opening.

[0065] Embodiment 7 A microcavity plate according to embodiment 4, further comprising a gasket material.

[0066] Embodiment 8 The gasket material is provided integrally with the bottom surface of the upper grid. The microcavity plate according to Embodiment 7, wherein when the upper grid is inserted into the open well, the gasket material is positioned between the bottom surface of the upper grid and the upper surface of the microcavity substrate.

[0067] Embodiment 9 The microcavity plate according to Embodiment 1, wherein the plurality of microcavities include 1536 individual microcavities.

[0068] Embodiment 10 The microcavity plate according to Embodiment 1, wherein each of the plurality of microcavities has a top surface and a rounded bottom surface.

[0069] Embodiment 11 The microcavity plate according to Embodiment 10, wherein the diameter of each microcavity on the upper surface of each microcavity is approximately 1500 μm.

[0070] Embodiment 12 The microcavity plate according to Embodiment 10, wherein the inner surface of the cavity is covered with a non-adherent film to cells.

[0071] Embodiment 13 The microcavity plate according to Embodiment 12, wherein the coating includes an ultra-low adhesion (ULA) surface coating.

[0072] Embodiment 14 The microcavity plate according to Embodiment 4, wherein the upper grid comprises a plurality of protrusions extending from the outer circumference of the upper grid.

[0073] Embodiment 15 The microcavity plate according to embodiment 14, wherein some of the multiple protrusions are configured to align with and interlock with a plurality of through holes provided in the side wall of the open well.

[0074] Embodiment 16 The microcavity plate according to Embodiment 1, wherein the microcavity substrate is formed from a polymer selected from polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polymethylpentene, polycarbonate, polymethyl methacrylate, styrene-butadiene copolymer, styrene-ethylene-butylene-styrene, other similar polymers, or combinations thereof.

[0075] Embodiment 17 The microcavity plate according to Embodiment 16, wherein the microcavity substrate is formed from polystyrene.

[0076] Embodiment 18 The microcavity plate according to Embodiment 1, wherein the base is formed from a polymer comprising polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polymethylpentene, polycarbonate, polymethyl methacrylate, styrene-butadiene copolymer, styrene-ethylene-butylene-styrene, other similar polymers, or combinations thereof.

[0077] Embodiment 19 The microcavity plate according to embodiment 18, wherein the base is formed from polystyrene.

[0078] Embodiment 20 The microcavity plate according to Embodiment 1, wherein the upper grid is formed from an elastomer material selected from natural rubber, styrene-butadiene block copolymer, styrene-ethylene-butylene-styrene polymer, polyisoprene, polybutadiene, ethylene propylene rubber, ethylene propylene diene rubber, silicone elastomer, fluoroelastomer, polyurethane elastomer, nitrile rubber, or a combination thereof.

[0079] Embodiment 21 The microcavity plate according to Embodiment 7, wherein the gasket material is formed from an elastomer.

[0080] Embodiment 22 The microcavity plate according to Embodiment 21, wherein the elastomer contains silicone.

[0081] Embodiment 23 The microcavity plate according to Embodiment 7, wherein the gasket material is formed from a pressure-sensitive adhesive.

[0082] Embodiment 24 The microcavity plate according to Embodiment 1, wherein the microcavity plate is used for cell culture of spheroids.

[0083] Embodiment 25 The microcavity plate according to Embodiment 1, wherein the microcavity plate is used for high-throughput screening.

[0084] Embodiment 26 A method for performing high-throughput screening, The step of seeding cells into a microcavity plate as described in Embodiment 1, The steps include culturing the cells to form spheroids in the plurality of microcavities, The steps include: attaching an upper grid to the upper part of the plurality of microcavities in the microcavity plate to form a microcavity well; The steps include: handling each of the microcavity wells individually and performing high-throughput screening of the cultured spheroids; Methods that include...

[0085] Embodiment 27 The method according to Embodiment 26, wherein the step of culturing the cells includes the step of bringing the cells in the microcavity plate into contact with a cell culture medium. [Explanation of Symbols]

[0086] 10, 303 base 11 Upper part of the base 12 Flange (skirt) 15 Multiple wells 17 Lower Grid 20 Microcavity Substrates 23 Microcavities 27. Bottom of the microcavity 30, 405 Top grid 31 Protrusion 33 Top of the top grid 35 Bottom of the upper grid 37, 411 Grid segments in the first direction 39, 413 Second direction (vertical direction) grid segments 50 microcavity wells 55 Sidewalls of each microcavity well 60 Gasket Sheets (Gasket Material) 61 Through hole 300, 500, 600, 700, 800 microcavity plates 307 Open Well 309 Sidewall of an open well 310 each microcavity 315 Multiple microcavities 320 rows of each microcavity 325 Multiple microcavity rows 330 Microcavity Rows 335 Multiple Microcavity Rows 375 Individual Microcavity Wells 417 Opening 420 each opening row 425 Multiple opening rows 430 Each row of openings 435 Multiple rows of openings 520 individual microcavity rows 525 Multiple individual microcavity rows 530 Individual microcavity rows 535 Multiple individual microcavity rows

Claims

1. A microcavity plate used for high-throughput screening of spheroid cell cultures, A lower grid comprising multiple grid segments that form multiple openings by being arranged in a regular matrix, An open well comprising a plurality of side walls extending vertically from the outer periphery of the lower grid, A base equipped with, A microcavity substrate comprising a plurality of microcavities, each having a cavity positioned within the openings of the lower grid, arranged in a regular matrix with respect to the plurality of openings of the lower grid, An upper grid having a plurality of regular matrix-shaped well openings that mirror the regular matrix of the lower grid, Equipped with, The upper grid is configured to be positioned on the lower grid that demarcates the regular matrix at the base within the open well, A microcavity plate in which the plurality of well openings are aligned with the plurality of microcavities of the microcavity substrate.

2. The microcavity plate according to claim 1, wherein a microcavity well is defined by a plurality of side walls composed of grid segments defining each microcavity opening, and an individual microcavity positioned at the center of the microcavity opening.

3. The microcavity plate further comprises a gasket material, The microcavity plate according to claim 1, wherein when the upper grid is inserted into the open well, the gasket material is positioned between the bottom surface of the upper grid and the upper surface of the microcavity substrate.

4. The microcavity plate according to claim 1, wherein the plurality of microcavities include 1,536 individual microcavities.

5. The microcavity plate according to claim 1, wherein each of the plurality of microcavities has a top surface and a rounded bottom surface.

6. The microcavity plate according to claim 5, wherein the diameter of each microcavity on the upper surface of each microcavity is 1500 μm.

7. The microcavity plate according to claim 5, wherein the inner surface of the cavity is coated with an ultra-low adhesion (ULA) surface coating.

8. The upper grid comprises a plurality of protrusions extending from the outer circumference of the upper grid, The microcavity plate according to claim 1, wherein the projection is configured to align with and interlock with a plurality of through holes provided in the side wall of the open well.

9. The microcavity plate according to claim 1, wherein each of the microcavity substrate and the base is formed from a polymer selected from polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polymethylpentene, polycarbonate, polymethyl methacrylate, styrene-butadiene copolymer, styrene-ethylene-butylene-styrene, other similar polymers, or combinations thereof.

10. The microcavity plate according to claim 9, wherein each of the microcavity substrate and the base is formed from polystyrene.

11. The microcavity plate according to claim 1, wherein the upper grid is formed from an elastomer material selected from natural rubber, styrene-butadiene block copolymer, styrene-ethylene-butylene-styrene polymer, polyisoprene, polybutadiene, ethylene propylene rubber, ethylene propylene diene rubber, silicone elastomer, fluoroelastomer, polyurethane elastomer, nitrile rubber, or a combination thereof.

12. The microcavity plate according to claim 3, wherein the gasket material is formed from an elastomer.

13. The microcavity plate according to claim 12, wherein the elastomer comprises silicone.

14. The microcavity plate according to claim 3, wherein the gasket material is formed from a pressure-sensitive adhesive.

15. A method for performing high-throughput screening, The step of seeding cells into a microcavity plate according to claim 1, The steps include culturing the cells to form spheroids in the plurality of microcavities, The steps include bringing the cells in the microcavity plate into contact with a cell culture medium, The steps include: handling each microcavity well individually and performing high-throughput screening of the cultured spheroids; Methods that include...

Images