Patent Application
20230383226
This disclosure generally relates to microcavity plates. In particular, the present disclosure relates to microcavity plates for use in cell culture and high throughput screening.
Drug discovery and development is a first step in the process for finding treatment methods for many diseases. That step includes testing many molecular compounds as potential candidates for medical treatment. Due to the amount of testing, high throughput screening (HTS) is often used to conduct the testing. HTS uses automated equipment to rapidly test biological activity of numerous samples. Microplates are often used for HTS, and a 1536-well plate is a typical configuration of microplate for which there is automated handling.
In order to more accurately represent the environment experienced by cells in vivo, 3D cell culture models or spheroids may be used in HTS. The spheroids or 3D cell cultures generally have diameters in the range of about 100-300 microns. Conventional technologies may allow spheroids to be dispensed in 96-well or 384-well plates. However, due to the size of the spheroids, the well geometry in 1536-well plates may not allow for dispensing spheroids into wells of a 1536-well plate. Therefore, conventional 1536-well cell culture devices for generating 3D cell cultures or spheroids typically do not lend themselves to automated handling in HTS processes. Furthermore, there is a lack of conventional equipment available to dispense large structures such as spheroids cultured in a bulk spheroid production vessel into 1536-well plates for HTS.
Embodiments of the disclosure provide a microcavity plate for bulk spheroid production that may also be used in HTS processes. The microcavity plate comprises 1536 shallow cavities with each cavity having a 1500 μm diameter. The cavities are arranged in the same ordered rows and columns as a typical 1536-well plate. In embodiments, a grid may be added to a top surface of the microcavities to treat each microcavity as an individual to permit HTS. Therefore, embodiments of the disclosure resolve the existing conventional equipment issues, since the spheroids cultured together from the start of the culture process can then be addressed individually after placement of the grid.
In an aspect, a microcavity plate comprises a base comprising: a bottom grid comprising a plurality of grid segments arranged in ordered rows and columns to form a plurality of openings, and an open well comprising a plurality of sidewalls extending vertically from a perimeter of the bottom grid; and a microcavity substrate comprising a plurality of microcavities arranged in ordered rows and columns that align with the plurality of openings in the bottom grid, each microcavity comprising a cavity disposed within an opening of the bottom grid.
In some embodiments, the microcavity plate further comprises an upper grid. In some embodiments, the upper grid comprises a plurality of well openings in ordered rows and columns that mirror the ordered rows and columns of the bottom grid. In some embodiments, the upper grid is configured for placement in the open well on top of the bottom grid delineating the ordered rows and columns of the base. In some embodiments, the plurality of well openings align with the plurality of microcavities in the microcavity substrate. In some embodiments, a microcavity well is defined by sidewalls comprising grid segments that define each microcavity opening, and an individual microcavity centered within the microcavity opening.
In some embodiments, the microcavity plate further comprises a gasket material. In some embodiments, the gasket material is integrated on a bottom of the upper grid, wherein when the upper grid is inserted in the open well, the gasket material is disposed between the bottom of the upper grid and a top of the microcavity substrate.
In some embodiments, the plurality of microcavities comprises 1536 individual microcavities.
In some embodiments, each cavity in the plurality of microcavities comprises a top surface and a rounded bottom. In some embodiments, a diameter of each microcavity at the top surface of the microcavity is about 1500 μm.
In some embodiments, an inner surface of the cavity is coated with a coating non-adherent to cells. In some embodiments, the coating comprises an ultra low attachment (ULA) surface coating.
In some embodiments, the upper grid comprises a plurality of protrusions extending from a perimeter of the upper grid. In some embodiments, protrusions in the plurality of protrusions are configured to align and interlock with a plurality of through holes in the sidewalls of the open well.
In some embodiments, the microcavity substrate is formed from polymers selected from polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polymethylpentene, polycarbonate, polymethyl methacrylate, styrene-butadiene copolymers, styrene-ethylene-butylene-styrene, other such polymers, or a combination thereof. In some embodiments, the microcavity substrate is formed from polystyrene.
In some embodiments, the base is formed from a polymer comprising polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polymethylpentene, polycarbonate, polymethyl methacrylate, styrene-butadiene copolymers, styrene-ethylene-butylene-styrene, other such polymers, or a combination thereof. In some embodiments, the base is formed from polystyrene.
In some embodiments, the upper grid is formed from elastomeric materials selected from natural rubbers, styrene-butadiene block copolymers, styrene-ethylene-butylene-styrene polymers, polyisoprene, polybutadiene, ethylene propylene rubber, ethylene propylene diene rubber, silicone elastomers, fluoroelastomers, polyurethane elastomers, nitrile rubbers, or a combination thereof.
In some embodiments, the gasket material is formed from an elastomer. In some embodiments, the elastomer comprises silicone. In some embodiments, the gasket material is a pressure sensitive adhesive.
In some embodiments, the microcavity plate is used for cell culture of spheroids. In some embodiments, the microcavity plate is used for high-throughput screening.
In an aspect, a method of high-throughput screening comprises seeding cells in a microcavity plate as described herein. The method further comprises culturing the cells to form spheroids within the plurality of microcavities; attaching an upper grid to a top portion of the plurality of microcavities within the microcavity plate to form microcavity wells; and treating each of the microcavity wells as an individual for high throughput screening of the cultured spheroids. In some embodiments, culturing the cells comprises contacting the cells in the microcavity plate with cell culture media.
In order to more accurately represent the environment experienced by cells in vivo, 3D cell culture models may be used in HTS. Recent research demonstrates that cell responses in 3D cultures, such as 3D spheroids or organoids (hereafter referred to as spheroids), are more similar to in vivo behavior than the cell responses in two-dimension (2D) cultures where cells are cultured in a monolayer. The additional dimensionality of 3D cultures is believed to lead to the differences in cellular responses because it influences the spatial organization of the cell surface receptors engaged in interactions with surrounding cells and induces physical constraints to cells, thereby affecting the signal transduction from the outside to the inside of cells, ultimately influencing gene expression and cellular behavior. Conventional culture devices for generating 3D cell cultures or spheroids do not optimally lend themselves to automated handling in HTS processes.
Microplates are often used for HTS, and a 1536-well plate is a typical configuration of microplate for which there is automated handling.
In contrast to microplate wells, which are millimeter (mm) or centimeter (cm)-sized, conventional culture devices for generating 3D cell cultures or spheroids have micron-sized wells. There are several different names for culture devices having wells that are micron-sized, including microcavities and microspaces. Microcavities typically include an inner cavity with a rounded bottom that is non-adherent to cells, and which facilitates 3D cell culture by allowing cells seeded into the microcavities to self-assemble or attach to one another to form a spheroid in each microcavity. Typical microcavities are shallow (˜500-3000 μm) and permit cell culture medium to cover all of the spheroids in all cavities at once to make manual handling easy.
Microcavity vessels are referred to as bulk spheroid production vessels and can culture many spheroids at once due to the micron-sized geometry of the wells and hexagonal close-packing density or “honeycomb” well configuration.
Embodiments of the disclosure provide a microcavity plate for bulk spheroid production that may also be used in HTS processes. The microcavity plate comprises 1536 shallow cavities with each cavity having a 1500 μm diameter. The cavities are arranged in the same ordered rows and columns as a typical 1536-well plate. The ordered arrangement of the microcavities in rows and columns allows for an upper grid to be added to a top surface of the microcavities. By adding the upper grid, each microcavity has its own well and can be treated as an individual to permit HTS. Therefore, embodiments of the disclosure allow for spheroids to be cultured together from the start of the culture process to create a homogeneous culture environment, but also allow spheroids to be addressed individually after placement of the grid.
Microcavity plates according to embodiments of the disclosure provide a homogenous culturing environment. Before an upper grid is disposed in the microcavity plate to form individual wells, all spheroids cultured in the 1536-well microcavity plate may receive the same treatment at the same time, thereby providing a homogenous culture environment. In contrast, typical plates with individual wells have more of a heterogenous culture environment because dispensing the same volume to each well is difficult, even with automated equipment.
Microcavity plates according to embodiments described herein may be converted into a plate with individual wells through the addition of an upper grid. The upper grid may be disposed within the plate at a top of the plurality of microcavities. The configuration of a microcavity plate with individual wells will allow for users to perform HTS, as the subsequently added grid will allow the spheroid in each well to be treated separately.
Microcavity plates of embodiments described herein do not require an automated dispenser for handling spheroids or transferring spheroids from a bulk spheroid production vessel to a 1536-well plate.
The microcavity substrate may be formed from a film. As a nonlimiting example, the microcavity substrate may be formed from a flat film having a thickness of 0.003-0.015 inches. The film may be formed of any suitable material, and nonlimiting examples include polystyrene, polymethylpentene, polyethylene, polypropylene, or laminates. A thickness at the rounded-bottom apex of the microcavity substrate may be in a range of about 35 microns to about 75 microns. Elsewhere, the thickness of the microcavity substrate may vary.
In some embodiments, the upper grid or top grid can be molded as a singular piece. In some embodiments, the bottom component can be molded as a singular piece, wherein the bottom component includes the base, bottom grid, and microcavity substrate. The gasket material may be overmolded on the top grid. In some embodiments, the bottom component may be formed by injection molding. The gasket material may be any suitable material, such as an elastomer. In some embodiments, the elastomer may be silicone. In some embodiments, the gasket material is a pressure sensitive adhesive.
The microcavity wells may have any suitable non-binding coating. For example, the coating may be a surface coating that is non-adherent to cells. In some embodiments, the cell non-adherent surface coating is a Corning Ultra Low Attachment (ULA) surface coating. The Corning ULA surface is hydrophilic, biologically inert and non-degradable, which promotes highly reproducible spheroid formation and easy harvesting. The covalent attachment of Ultra-Low Attachment surface reduces cellular adhesion to the well surface. The Ultra-Low Attachment (ULA) surface allows for uniform and reproducible 3D multicellular spheroid formation. The 1536 microcavity well format allows for high throughput 3D cell culture and analysis.
Microcavity plates according to embodiments described herein may be formed of any suitable material. In some examples, the plate may be formed in steps. For example, one step may be to construct the bottom component of the 1536 microcavity plate, wherein the bottom component comprises the base of the plate and the bottom grid portion. The material of construction may comprise a plastic polymer, co-polymer, or polymer blend. Nonlimiting examples include polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polymethylpentene, polycarbonate, polymethyl methacrylate, styrene-butadiene copolymers, styrene-ethylene-butylene-styrene, other such polymers, or a combination thereof. Any suitable construction method may be used to form the bottom grid or base of the microplate. Nonlimiting examples include injection molding, thermoforming, 3D printing, or any other method suitable for forming a plastic part.
The microcavity substrate may be formed at the same time as a bottom grid portion. The microcavity substrate may be formed from the same material or a similar material and method for making the rest of the plate. In some embodiments, the microcavity substrate may be molded or formed separately from the rest of the plate and bonded subsequently through thermal-bonding, ultrasonic welding, or any other method of plastic joining. The material of construction for the microcavity substrate may comprise a plastic polymer, co-polymer, or polymer blend. Nonlimiting examples include polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polymethylpentene, polycarbonate, polymethyl methacrylate, styrene-butadiene copolymers, styrene-ethylene-butylene-styrene polymers, other such polymers, or a combination thereof. Any suitable construction method may be used to form the microcavity substrate, such as nonlimiting examples include injection molding, thermoforming, 3D printing, or any other method suitable for forming a plastic part.
The upper grid may be formed using similar materials as the base or bottom grid and the microcavity substrate. In some embodiments, the upper grid is formed using more elastomeric materials such as natural rubbers, styrene-butadiene block copolymers, styrene-ethylene-butylene-styrene polymers, polyisoprene, polybutadiene, ethylene propylene rubber, ethylene propylene diene rubber, silicone elastomers, fluoroelastomers, polyurethane elastomers, and nitrile rubbers. Any suitable construction method may be used to form the upper grid of the microplate, such as nonlimiting examples include injection molding, thermoforming, 3D printing, or any other method suitable for forming a plastic part.
In some embodiments, the manufacture of the upper grid comprises an additional process to add an elastomer to the portion of the grid that will directly contact the substrate, if the upper grid is made from a non-elastomeric material. The elastomer facilitates a seal between the substrate and the upper grid to maintain the integrity of individual microcavity wells once the upper grid is in place. In such embodiments, the upper grid would be supplied separated from the microcavity plate and in its own packaging, as the upper grid would need to be kept sterile while the rest of the plate is used for cell culture.
According to embodiments of the present disclosure, methods for culturing cells or capturing cells on microcavity plate as described herein are also disclosed. In some embodiments, methods comprise cell culture of cell aggregates, or spheroids, in microcavity plate.
Embodiments of the disclosure further comprise methods of using a microcavity plate described herein. To use the 1536 microcavity plate, a user would make sure the upper grid is not within the microcavity plate before seeding cells in the microcavity plate. This would allow for ease of manual handling and maintenance of an initially homogenous culture environment. Once the culture has formed the required characteristics, such as number of cells or spheroids, differentiated state, etc., the cell culture medium in the plate would be removed. Note that the cell culture medium would be removed for the most part, as some cell culture medium would naturally remain in the individual microcavities with the spheroids. At that point, the upper grid could then be inserted, and medium or reagents may be added to perform HTS.
In an embodiment, a method of high-throughput screening comprises seeding cells in a microcavity plate; culturing the cells to form spheroids within the plurality of microcavities; attaching an upper grid to a top portion of the plurality of microcavities within the microcavity plate to form microcavity wells; and treating each of the microcavity wells as an individual for high throughput screening of the cultured spheroids. Culturing the cells may comprise contacting the cells in the microcavity plate with cell culture media.
Any type of cells may be cultured on the microcavity plate including, but not limited to, immortalized cells, primary culture cells, cancer cells, stem cells (e.g., embryonic or induced pluripotent), etc. The cells may be mammalian cells, avian cells, piscine cells, etc. The cells may be of any tissue type including, but not limited to, kidney, fibroblast, breast, skin, brain, ovary, lung, bone, nerve, muscle, cardiac, colorectal, pancreas, immune (e.g., B cell), blood, etc. The cells may be in any cultured form including disperse (e.g., freshly seeded), confluent, 2-dimensional, 3-dimensional, spheroid, etc.
Culturing cells on a microcavity plate may include seeding cells on the microcavity plate. Seeding cells on a microcavity plate may include contacting the plate with a solution containing the cells. Culturing cells on microcavity plate may further include contacting the microcavity plate with cell culture medium. Generally, contacting the microcavity plate with cell culture medium includes seeding or placing cells to be cultured on the microcavity plate in an environment with medium in which the cells are to be cultured. Contacting the microcavity plate with cell culture medium may include pipetting cell culture medium onto the microcavity plate. In some embodiments, cell culture medium may be disposed in the plate for a predetermined period of time, at least some of the cell culture medium may be removed after the predetermined period of time, and fresh cell culture medium may be added. Cell culture medium may be removed and replaced according to any predetermined schedule. For example, at least some of the cell culture medium may be removed and replaced every hour, or every 12 hours, or every 24 hours, or every 2 days, or every 3 days, or every 4 days, or every 5 days.
Any cell culture medium capable of supporting the growth of cells may be used. Cell culture medium may be for example, but is not limited to, sugars, salts, amino acids, serum (e.g., fetal bovine serum), antibiotics, growth factors, differentiation factors, colorant, or other desired factors. Exemplary cell culture medium includes Dulbecco's Modified Eagle Medium (DMEM), Ham's F12 Nutrient Mixture, Minimum Essential Media (MEM), RPMI Medium, Iscove's Modified Dulbecco's Medium (IMDM), MesenCult™-XF medium (commercially available from STEMCELL Technologies Inc.), and the like.
It will be appreciated that the various disclosed embodiments may involve particular features, elements, or steps that are described in connection with that particular embodiment. It will also be appreciated that a particular feature, element, or step, although described in relation to one particular embodiment, may be interchanged or combined with alternate embodiments in various non-illustrated combinations or permutations.
It is also to be understood that, as used herein the terms “the,” “a,” or “an,” mean “at least one,” and should not be limited to “only one” unless explicitly indicated to the contrary. Thus, for example, reference to “an opening” includes examples having two or more such “openings” unless the context clearly indicates otherwise.
All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.
As used herein, “have,” “having,” “include,” “including,” “comprise,” “comprising,” or the like are used in their open-ended sense, and generally mean “including, but not limited to.”
Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, examples include from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
All numerical values expressed herein are to be interpreted as including “about,” whether or not so stated, unless expressly indicated otherwise. It is further understood, however, that each numerical value recited is precisely contemplated as well, regardless of whether it is expressed as “about” that value. Thus, “a dimension less than 10 mm” and “a dimension less than about 10 mm” both include embodiments of “a dimension less than about 10 mm” as well as “a dimension less than 10 mm.”
Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that any particular order be inferred.
While various features, elements or steps of particular embodiments may be disclosed using the transitional phrase “comprising,” it is to be understood that alternative embodiments, including those that may be described using the transitional phrases “consisting” or “consisting essentially of,” are implied. Thus, for example, implied alternative embodiments to a method comprising A+B+C include embodiments where a method consists of A+B+C, and embodiments where a method consists essentially of A+B+C.
Although multiple embodiments of the present disclosure have been described in the Detailed Description, it should be understood that the disclosure is not limited to the disclosed embodiments, but is capable of numerous rearrangements, modifications and substitutions without departing from the disclosure as set forth and defined by the following claims.
This application claims the benefit of priority under 35 U.S.C. § 119 of U.S. Provisional Application Ser. No. 63/107,663 filed on Oct. 30, 2020, the content of which is relied upon and incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/056736 | 10/27/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63107663 | Oct 2020 | US |
