JP2021072811A - Device and method for generation and culture of 3d cell aggregate - Google Patents

Abstract

To provide devices and methods for generation and culture of 3D cell aggregates.SOLUTION: A substrate is provided that contains or comprises an array of microwells or wells. The substrate can form a part of a multiwell plate, a flask, a dish, a tube, a multi-layer cell culture flask, a bioreactor, or any other laboratory container intended to grow cells or spheroids. The microwells or wells provide an environment that is conducive to the formation of spheroids in culture and have spheroid-inducing geometry.SELECTED DRAWING: Figure 8

Description

Cross-reference of related applications

This application is incorporated herein by reference in its entirety, US Provisional Patent Application No. 62/072015, filed October 29, 2014, and October 29, 2014, the same No. 62 /. Claims the priority of No. 072103, No. 62/072088 filed on October 29, 2014, and No. 62/094471 filed on December 19, 2014.

The present disclosure relates to devices, systems, and methods for culturing cells. In particular, devices and methods for the production and culture of 3D cell aggregates are provided.

Three-dimensional (3D) cell culture is the proliferation of cells in an artificially created environment that allows cells to proliferate and / or interact primarily with each other in all three dimensions. 3D cell culture exhibits an improvement over the way cells grow in 2D (eg, on a Petri dish), at least because 3D conditions more accurately embody the in vivo environment.

Cells cultured in three dimensions, such as spheroids, may exhibit even more in vivo-like functions than their counterparts cultured in two dimensions as a monolayer. In a two-dimensional cell culture system, cells can attach to the substrate on which they are cultured. However, when cells grow three-dimensionally, such as spheroids, the cells interact with each other rather than attach to the substrate. One of the problems with spheroid-based assays is that the results of the assay typically vary with the size of the spheroid. For example, variations in variables such as seeding density and growth time from system to system can affect assay reproducibility on a system-by-system or well-by-well basis within a given system. Therefore, maintaining consistent spheroid dimensions between spheroids grown in separate wells can present challenges.

As the density of cells growing in the cell culture apparatus increases, a larger volume of cell culture medium, or more frequent replacement of the cell culture medium, may be required to maintain the cells. However, increasing the frequency of medium changes can be inconvenient. In addition, an increase in the volume of the cell culture medium can lead to an undesired increase in the height of the medium above the cells to be cultured. As the height of the medium increases, the rate of gas exchange for cells through the medium decreases.

Cells are densely proliferated as spheroid masses in wavy bags, spinners and shakers. However, the dimensions of spheroids grown in such devices are inconsistent, and shearing specific to such devices tends to break the spheroids into smaller masses. Moreover, such devices will not be able to achieve cell densities high enough to meet actual demand.

The present disclosure relates to devices, systems and methods for culturing cells. In particular, devices and methods for the production and culture of 3D cell aggregates are provided. For example, devices and methods are provided to address problems known or unknown in the art that are detrimental to 3D culture of cells.

Cells cultured in three dimensions, such as spheroids, may exhibit higher in vivo-like function than their counterparts cultured in two dimensions as a monolayer. In a two-dimensional cell culture system, cells can attach to the substrate on which they are cultured. However, when cells grow three-dimensionally, such as spheroids, the cells interact with each other rather than attach to the substrate. Cells cultured in three dimensions resemble in vivo tissues in terms of cell-cell communication and extracellular matrix expression. Thus, spheroids provide an excellent model for cell migration, differentiation, survival, and proliferation, and thus provide a better system for research, diagnosis, and testing of efficacy, pharmacology, and toxicity.

In some embodiments, a substrate comprising or containing a microwell or an array of wells is provided. The substrate can form part of a cell culture device or device. For example, the substrate can form part of a multi-well plate, flask, dish, tube, multi-layer cell culture flask, bioreactor, or other research vessel intended for cell or spheroid growth. Microwells or wells (the terms "microwell" and "well" are used interchangeably in the present disclosure) are structured and arranged to provide an environment that facilitates the formation of spheroids in culture. That is, in embodiments, the microwells have a spheroid-inducing geometry. In addition, the wells are structured and arranged to allow the liquid to move in and out of the wells without trapping air between the substrate and the liquids or droplets taken into the wells. That is, in embodiments, the microwells have a capillary structure. For example, the wells in which the cells grow can be non-adhesive to the cells so that the cells in the wells associate with each other to form spheres. The spheroid extends to the dimensional tolerance imposed by the well geometry. In some embodiments, the wells are coated with an ultra-low binding material to make the wells non-adhesive to the cells.

In some embodiments, the cell culture device has a frame that includes the footprint of the device, and the substrate of the device is configured such that the cells cultured in the device form spheroids. There is. For example, the cell culture substrate in the device is non-adhesive to the cells so that the cells associate with each other instead of the substrate. The cell culture substrate is further composed of multiple microwells (or wells) that, due to their geometry, allow cells grown in the wells to form cell aggregates or spheroids of similar size. Spheroids extend to the dimensional tolerances imposed by the geometry of the microwells. In some embodiments, the wells have a hypobinding treatment or are coated with an ultra-low binding material to make the wells non-adhesive to cells.

Examples of non-adhesive materials include perfluoropolymers, olefins, or similar polymers, or mixtures thereof. Other examples include nonionic hydrogels such as agarose and polyacrylamide, polyols such as polyethers such as polyethylene oxide and polyols such as polyvinyl alcohol, or similar materials, or mixtures thereof. For example, a combination of non-adhesive wells, well geometry (eg, dimensions and shape), and / or gravity induces cells cultured in the wells to self-assemble into spheroids. Some spheroids maintain differentiated cell function with a more in vivo-like response compared to cells grown in a single layer. Other cell types, such as mesenchymal stromal cells, retain their pluripotency when cultured as spheroids.

In some embodiments, the systems, devices, and methods described herein include one or more cells. In some embodiments, the cells are stored frozen. In some embodiments, the cells are in a three-dimensional culture. In some such embodiments, the system, device, and method comprises one or more spheroids. In some embodiments, one or more cells actively divide. In some embodiments, the system, apparatus, and method comprises a culture medium (eg, nutrients (eg, proteins, peptides, amino acids), energy (eg, carbohydrates), essential metals and minerals (eg, calcium, magnesium, iron). , Phosphates, sulfates), buffers (eg phosphates, acetates), pH change indicators (eg phenol red, bromocresol purple), selective agents (eg chemicals) , Anti-microbial agents), etc.) are included. In some embodiments, one or more test compounds (eg, drugs) are included in the system, device, and method.

A wide variety of cell types can be cultured. In some embodiments, the spheroid comprises a single cell type. In some embodiments, the spheroid comprises two or more cell types. In some embodiments, when two or more spheroids are grown, each spheroid is of the same type, but in other embodiments, two or more different types of spheroids are grown. A cell proliferated in a spheroid can be a natural cell or a modified cell (eg, a cell containing one or more non-naturally modified genes). In some embodiments, the cell is a somatic cell. In some embodiments, the cell is a stem or progenitor cell (eg, pluripotent, multi-potent), fate-determined, immortalized, etc., in a desired state of differentiation. Embryonic stem cells, induced pluripotent stem cells). In some embodiments, the cells are disease cells or disease model cells. For example, in some embodiments, the spheroid comprises one or more types of cancer cells or cells that can be induced into a hyperproliferative state (eg, transformed cells). Cells may be derived from or derived from any desired tissue or organ type, adrenal gland, bladder, blood vessels, bones, bone marrow, brain, cartilage, cervix, corneal gland, endometrial membrane. , Esophageal, gastrointestinal, immune system (eg, T lymphocytes, B lymphocytes, white blood cells, macrophages, and dendritic cells), liver, lungs, lymph glands, muscles (eg, myocardium), nerves, ovaries, pancreas (eg, eg) Pancreatic islet cells), pituitary glands, prostate, adrenal glands, salivary glands, skin, tendons, testis, and thyroid, but not limited to them. In some embodiments, the cell is a mammalian cell (eg, human, mouse, rat, rabbit, dog, cat, cow, pig, chicken, goat, horse, etc.).

Cultured cells have been found to be used in a wide variety of research, diagnostics, drug screening and testing, therapeutic and industrial applications.

In some embodiments, cells are used for the production of proteins or viruses. Systems, devices, and methods for culturing a large number of spheroids in parallel are particularly effective for protein production. Three-dimensional culture allows for increased cell density per square centimeter of cell proliferation surface area and increased protein yield. The desired protein or virus for vaccine production can be propagated intracellularly and can be isolated or purified for the desired use. In some embodiments, the protein is a natural protein for the cell. In some embodiments, the protein is non-natural. In some embodiments, the protein is expressed recombinantly. Preferably, the protein is overexpressed using a non-natural promoter. The protein may be expressed as a fusion protein. In some embodiments, a tag for purification or detection is expressed as a fusion partner of the protein of interest to facilitate its purification and / or detection. In some embodiments, the fusion is expressed with a cleaving linker to allow separation of the fusion partner after purification.

In some embodiments, the protein is a therapeutic protein. Such proteins include molecules or presents that replace deficient or abnormal proteins (eg, insulin), enhance existing pathways (eg, inhibitors or agonists), and provide novel functions or activities. Proteins and peptides that interfere with the aircraft or deliver other compounds or proteins (eg, radionuclides, cytotoxic drugs, effector proteins, etc.), but are not limited thereto. In some embodiments, the protein is an immunoglobulin, such as an antibody of any kind (eg, humanized, bispecific, multispecific, etc.) (eg, a monoclonal antibody). The therapeutic proteins are classified as antibody-based drugs, Fc fusion proteins, anticoagulants, antigens, blood factors, bone morphogenetic proteins, modified protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents. However, it is not limited to them. Therapeutic proteins can be used to prevent or treat cancer, immune disorders, metabolic disorders, inherited genetic disorders, infectious diseases, and other diseases and conditions.

In some embodiments, the protein is a diagnostic protein. Diagnostic proteins include, but are not limited to, antibodies, affinity binding partners (eg, receptor binding ligands), inhibitors, antagonists and the like. In some embodiments, the diagnostic protein is, or is expressed with, a detection moiety (eg, a fluorescent moiety, a luminescent moiety (eg, luciferase), a colorimetric moiety, etc.).

In some embodiments, the protein is an industrial protein. Examples of industrial proteins include, but are not limited to, food components, industrial enzymes, agricultural proteins, analytical enzymes, and the like.

In some embodiments, cells are used for drug discovery, characterization, efficacy testing, and toxicity testing. Such tests include pharmacological effect evaluation, carcinogenicity evaluation, medical contrast agent characteristic evaluation, half-life evaluation, radiation safety evaluation, genetic toxicity test, immunotoxicity test, reproduction and development test, drug interaction evaluation, and dose evaluation. , Adsorption evaluation, pharmacokinetic evaluation (disposition assessment), metabolism evaluation, removal research, etc., but not limited to them. Specific cell types can be used in specific tests (eg, hepatocytes for hepatotoxicity, proximal renal tubule epithelial cells for nephropathy, vascular endothelial cells for vascular toxicity, nerve cells for neurotoxicity and Glial cells, myocardial cells for cardiotoxicity, skeletal muscle cells for collateral lysis). Treated cells can be evaluated for any desired parameter including, but not limited to, membrane integrity, intracellular metabolite content, mitochondrial function, lysosomal function, apoptosis, gene mutations, differences in gene expression, and the like.

In some embodiments, the cell culture apparatus is a component of the larger system. In some embodiments, the system is a plurality of such cell culture devices (eg, 2, 3, 4, 5, ..., 10, ..., 20, ..., 50, ..., 100, ..., 1000, etc.). Includes. In some embodiments, the system comprises an incubator to maintain the incubator under optimal culture conditions (eg, temperature, atmosphere, humidity, etc.). In some embodiments, the system comprises a detector for imaging or otherwise analyzing cells. Such detectors include fluorescence photometers, luminometers, cameras, microscopes, plate readers (eg, EnVision plate readers from PERKIN ELMER; ViewLux plate readers from PERKIN ELMER), cell analyzers (eg, GE). IN cell analyzers 2000 and 2200; THERMO's CELLOMICS CELLNSIGHT high-content screening platform) and confocal imaging systems (eg, PERKIN ELMER's OperaPhenix high-throughput content screening system; GE's INCELL6000 cell imaging system). However, it is not limited to them. In some embodiments, the system comprises a perfusion system or other component for supplying, resupplying, and circulating the culture medium or other components to the cultured cells. In some embodiments, the system comprises robotic components (eg, pipettes, arms, plate movers, etc.) for automating the handling, use, and / or analysis of the incubator.

Ensure complete elimination of air from the microwells during the introduction of aqueous liquids in the handling of microwell-formatted microplates or other containers with microwells, and especially when adding liquids to the microwells. You should be careful about what you do. When adding a liquid to a container containing the wells, air can be trapped under the liquid and in the wells (eg, microwells), especially if the wells have a circular cross section. Since the surface tension of the aqueous liquid applied to the wells is strong, the droplets tend to retain a spherical shape. Spherical droplets can easily block circular holes of similar size, trapping air in the holes (eg, microwells).

In some embodiments, the geometry of the wells, which reduces the likelihood of air being trapped in the microwells, while maintaining the cell culture properties of the wells (eg, usefulness in 3D cell culture), is described herein. Provided at. In some embodiments, the geometry of the wells (eg, the geometry of the microwells) allows for the efficient removal of air during the introduction of liquid into the wells. In some embodiments, the geometry of the wells provides a path for the flow of liquid into the wells without blocking the escape of air from the wells. In some embodiments, the geometry of the wells provides a path for the trapped air to escape. In some embodiments, the various wells maintain a confinement dimension for cell aggregation while at the same time facilitating the elimination of air within the microwells and allowing the influx of liquid into the microwells. Geometric shapes are provided herein.

In embodiments, the disclosure provides, for example, a device for culturing and evaluating spheroid cell masses or other aggregated cell populations (eg, multi-well plates, Petri dishes, flasks, multi-layer flasks, or HyperStack). In some embodiments, the device has at least one chamber (eg, well (eg, well)) with an opening (eg, an opening), one or more side walls, and a bottom surface having one or more microwells. , Macrowell), flask, etc.). In some embodiments, the geometry of the openings, sidewalls, and bottom is a 3D culture of cells (eg, cell aggregates, spheroids, etc.) in a chamber, as well as one or more of the following (eg, spheroids, etc.). (Everything) is possible: (1) Elimination of air from the chamber during distribution of the reagent (eg, liquid reagent) to the chamber (eg, without air being trapped under or in the liquid), (2). ) Routes of liquid flow to the microwells that reduce the possibility of air trapping under the surface of the liquids in the microwells, (3) Routes for air bleeding during the introduction of liquids into the microwells And / or (4) Paths for the escape of air trapped under the liquid surface.

In some embodiments, the device comprises a well (eg, a microwell) and a surface containing one or more wells (eg, a microwell) whose surface does not have a polygonal angle (eg, an angle of 90 degrees). Prepared with.

In some embodiments, the wells (eg, microwells) have a cross-sectional shape that resembles a sine wave. In such an embodiment, the bottom of the well is rounded (eg, hemispherical), the sidewalls increase in diameter from the bottom to the top of the well, and the boundaries between the wells are rounded. Thus, the top of the well does not terminate at a right angle. In some embodiments, the well has a diameter D (also referred to as the _{middle D) at the midpoint between the bottom and top, a diameter D top at} the top of the well, and a height H from the bottom to the top of the well. Have. In these embodiments, the _{upper part} of D is larger than D. Both relative and absolute dimensions of the wells can be selected for the desired culture conditions. For spheroid growth, the diameter D is preferably 1-3 times the desired diameter of the 3D cell aggregates cultured in the wells. The height H is 0.7 to 1.3 times D. _{The upper part} of the diameter D is 1.5 to 2.5 times D. D is preferably in the range of 100 micrometers (μm) to about 2000 micrometers (range between any two of the following values (eg, 200 to 1000 μm, 200 to 750 μm, 300 to 750 μm,). 400-600 μm, etc.), eg, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, or 2000 micrometers. ). However, alternative relative or absolute dimensions may be used. For example, D is 1 to 10 times the desired diameter of the cell aggregate (eg, 2, 3, 4, 5, 6, 7, 8, 9), or any value or range between them (eg, 2), 3, 4, 5, 6, 7, 8, 9). 1, 1 to 1.5, 1 to 2, 2, 1 to 2.5, 1 to 3, 2 to 3, 1 to 5, 3 to 5, 2 to 7 and the like). D is 100 μm to 10,000 μm, or any value (eg, 100, 200, 500, 1000, 2000, 5000) or range (eg, 100-2000, 200-1000, 300-700,) between them. 400-600, 500, etc.). H is 0.5 to 10 times D (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, or any value or range between them). The _{upper part} of D is 1.1 to 5 times that of D (for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9). , 2, 3, 4, 5, or any value or range between them).

Adjacent wells (eg, microwell) barrier between the the adjoining wells may have a reverse of the same shape, it may have a larger or smaller diameter D _B, or another method (For example, the shape of the bottom of the well may be different from the shape of the top of the well / barrier, see, for example, FIG. 2). To maximize the number of wells in a given surface, D _B is preferably less than D. D _B is 1.1 to 5 times greater than D (e.g., 1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9 , 2, 3, 4, 5, or any value or range between them), which may be large or small.

In certain embodiments, the cell culture apparatus herein comprises a plurality of wells, each configured such that cells cultured in the wells form spheroids of a particular diameter. The cell culture apparatus may include a structure that defines a plurality of wells. In some embodiments, each of the plurality of wells defines a top opening, a well bottom, and a side wall surface extending from the top opening to the well bottom. The side wall surface defines the nib area between the top opening and the bottom of the well. The cell culture volume is defined by the bottom surface, a portion of the side wall surface, and the nib region. In some embodiments, the nib region is in the range of 50 micrometers to 700 micrometers (eg, 50 μm, 75 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, and any range between them. ) From the bottom of the well, by diameter dimensions in the range of 100 micrometers to 700 micrometers (eg, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, and any range between them). Made. The sidewall surface or cell culture volume is sized to control the size of the spheroids that grow in each well. The nib area has a geometric shape that induces spheroids.

Embodiments include: for example: no uptake of air bubbles during cell seeding or medium exchange, high retention of 3D cell aggregates during medium exchange, ease of collection of spheroids from large area surfaces, gas. It provides many features, including permeability, medium reservoirs above multiple wells, spheroid-containing wells, and / or the ability to produce spheroids in large quantities and in uniform dimensions.

In some embodiments, the wells described herein comprise one or more capillary structures (eg, ridges, crevices, corners, acute angles, corrugations, pillars, etc.) extending from an upper opening within the wells. This capillary structure may extend from the top opening to the bottom of the well or from the top opening to the bottom of the mouth, which provides a route for air to escape as liquid flows into the well. To do. Suitable well geometries within the scope of the embodiments described herein include: (a) upper opening in a square cross section, rounded (eg, concave). A well with a well bottom and a side wall transitioning from a square cross section at the top of the well to a circular cross section at the bottom of the well; (b) from a top opening (eg, a top opening in a circular cross section) to a well bottom (eg, a circular cross section). A well with one or more protruding ridge lines extending to a rounded (eg, concave) well bottom (eg, see FIG. 1B); (c) Top opening (eg, top opening in a circular cross section). Wells with one or more fissures extending from the portion to the bottom of the well (eg, the bottom of a rounded (eg, concave) well (eg, see FIG. 2B); (d) do not completely surround the well. A well having a round bottom, a side wall completely surrounding the well, and a lower part of the well (see, eg, FIG. 27 or 28); (e) 1 A well (see, eg, FIG. 5), wherein one or more side walls have a convex cross section, thereby creating a sharp angle between the two side walls that can be formed by the pillars. In some embodiments, the wells are. Includes variations and / or combinations of the geometric shapes.

In embodiments, a method of making a cell culture apparatus with the wells described herein is provided herein.

In embodiments, for example, a method of using a cell culture device with a well described herein in a spheroid cell culture or cell assay is provided herein.

In some embodiments, a cell culture apparatus with a frame having wells located therein is provided herein in which the wells are (a) an upper opening; (b) rounded. Well bottom with cross-sectional shape; (c) one or more side walls extending from the well bottom to the top opening; (d) mouth as needed; and (e) below the surface of the liquid as needed It has a capillary structure that facilitates the introduction of liquid into the wells and the escape of air from the wells without forming a persistent air pool.

In some embodiments, the bottom of the well has a circular cross-section, where the top opening has a polygonal cross-section, and the side walls transition from a circular to a polygonal cross-section. This creates corners between the side walls that act as capillary structures that facilitate the introduction of liquid into the wells and the escape of air from the wells. In some embodiments, the transition of the cross-sectional shape of the sidewall does not impede the flow of fluid in and out of the well. In some embodiments, the top opening has a square or hexagonal cross-sectional shape.

In some embodiments, the structural feature that facilitates the introduction of liquid into the well and the escape of air from the well is a ridge that projects from one or more side walls and extends from the bottom of the well to the top opening. In some embodiments, the wells project from one or more side walls and extend from the bottom of the wells to the top opening, or approximately 1 to 20 ridges of such shape (eg, 1, 2, 3, 4). , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). In some embodiments, the bottom of the well has a circular cross-sectional shape and the top opening has a circular or polygonal cross-sectional shape. In some embodiments, the ridges are symmetrically spaced around the perimeter of the well. In some embodiments, the ridges are asymmetrically spaced around the perimeter of the well. In some embodiments, the ridge does not cover the entire distance from the top opening to the bottom of the well.

In some embodiments, the structural feature that facilitates the introduction of liquid into the well and the escape of air from the well is a fissure that extends within one or more sidewalls and from the bottom of the well to the top opening. In some embodiments, the wells have 1 to 20 fissures (eg, 1, 2, 3, 4, 5, 6, 7, 8,) extending within one or more sidewalls and from the bottom of the well to the top opening. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). In some embodiments, the bottom of the well has a circular cross-sectional shape and the top opening has a circular or polygonal cross-sectional shape. In some embodiments, the crevices are symmetrically spaced around the perimeter of the well. In some embodiments, the crevices are asymmetrically spaced around the perimeter of the well. In some embodiments, the crevice does not cover the entire distance from the top opening to the bottom of the well.

In some embodiments, the wells are defined by three or more adjacent pillars (eg, 3, 4, 5, 6, 7, 8, 9, 10), with a portion of the side of each pillar , Forming a side wall of the well; where the limited space between adjacent pillars forms a structural feature that facilitates the introduction of liquid into the well and the escape of air from the well. In some embodiments, the bottom of the well has a circular cross-sectional shape and extends below the pillars.

In some embodiments, a cell culture apparatus comprising a frame with a plurality of wells arranged therein is provided herein; where the wells are arranged in at least a row; the rows are: The gaps between the sidewalls are defined by two wavy sidewalls that are aligned so that they expand and narrow with each waveform; the top of each well is fluidized so that the tops of adjacent wells communicate with each other. It is defined by an enlarged gap between the two narrowed gaps in the side wall; the bottom of each well extends below the wavy side wall to form a circular well bottom. In some embodiments, the device comprises multiple rows of wells.

In some embodiments, one side wall, multiple side walls, and / or the bottom of the well are gas permeable and liquid impermeable. That is, in some embodiments, the substrate forming the well is gas permeable and liquid impermeable.

In some embodiments, one or more side walls are opaque and the bottom of the well is transparent.

In some embodiments, the well bottom comprises a concave arched surface.

In some embodiments, one side wall, multiple side walls, and / or the bottom of the well comprises a low adhesive or non-adhesive material and / or is coated with a low adhesive or non-adhesive material. To.

In some embodiments, the cell culture apparatus has 8 to about 10,000 wells (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, the surface with the microwell pattern is introduced into a wide range of cell culture products. In some embodiments, the bottom of the wells (eg, macrowells) in, for example, 12-, 24-, and 6-well plates, is patterned with a microwell surface. In some embodiments, the microwell surface is introduced into large surface area cell culture vessels such as T25, T75, T125, T175 and T250 flasks, as well as products from the CellSTACK and HYPERTack lines. In some embodiments, culturing cells in a large surface vessel with a large number of microwells is a large amount of 3D cell coagulation applicable for cell therapy applications, clonal cultures, stem cell niches, or co-cultures of niche cells. Produces aggregates.

In some embodiments, the cell culture apparatus herein is a bottom plate defining a main surface, one or more side walls extending from the bottom plate defining a reservoir, and a plurality formed on the main surface. Equipped with wells. Each well defines an upper opening that is coplanar with the main surface and open to the reservoir, and a nadir at the bottom of the well that is positioned below the main surface. In contrast to conventional well plates, the plates described herein define a reservoir above the surface of the well, which allows an increase in the volume of cell culture medium used, and thus frequently. Less medium exchange. See, for example, FIG.

In various embodiments, cell culture devices with one or more cell culture compartments are described. In some embodiments, the cell culture compartments are laminated. In some embodiments, each cell culture compartment comprises a substrate that defines a structured surface that defines multiple gas permeable wells. In some embodiments, the wells gas communicate with the outside of the device either directly through a gas permeable material or through vents or conduit spaces. In some embodiments, at least in part, a cell culture apparatus is described in which the wells have a substrate having an array of microwells made of a gas permeable material. Therefore, in some embodiments, the device puts a height of cell culture medium onto the cultured cells, which would be too high for existing cell culture devices, for efficient exchange of metabolic gases. Used to culture cells in the well while having. Since cells are cultured in gas-permeable wells that communicate with the outside of the device, gas exchange takes place through the wells and gas exchange through the cell culture medium due to the height of the medium above the cells. Overcome the shortage.

In certain embodiments, the cell culture apparatus comprises one or more cell culture compartments. In some embodiments, each cell culture compartment has an interior and comprises a substrate having a first main surface and a second main surface on the opposite side; the first main surface is a compartment. Define the structured surface inside the cell. In some embodiments, the structured surface defines multiple gas permeable wells. In some embodiments, the wells are in gas communication with the outside of the device.

In some embodiments, a method of culturing spheroids is provided herein comprising the step of filling the cell culture apparatus described herein with a culture medium; and the step of adding spheroid-forming cells to the culture medium. To. In some embodiments, the method further comprises the step of substituting / exchanging the medium (eg, daily, continuously, etc.).

In some embodiments, the use of the cell culture apparatus described herein for culturing spheroids is provided herein.

Additional features and advantages of the subject matter of the present disclosure are described in the detailed description below, some of which will be readily apparent to those skilled in the art or the following detailed description, claims, and attachments. Will be recognized by implementing the subject matter of the disclosure described herein, including the drawings of.

Both the above overview and the detailed description below present embodiments of the subject matter of the present disclosure and provide an overview or an overview to understand the nature and characteristics of the subject matter of the present disclosure as set forth in the claims. It should be understood that it is intended to provide a framework. The accompanying drawings are included to provide a better understanding of the subject matter of this disclosure and are incorporated herein by part. The drawings illustrate various embodiments of the subject matter of the present disclosure and serve to illustrate the principles and operations of the subject matter of the present disclosure as well as their description. In addition, the drawings and descriptions are intended to be merely exemplary and are not intended to limit the claims in any way.

Schematic of an exemplary embodiment of an array of wells 100. FIG. 1A is a cross-sectional view. Schematic of an exemplary embodiment of an array of wells 100. FIG. 1B is a top view of an exemplary embodiment of an array of wells cut along line BB of FIG. 1A. Schematic of another exemplary embodiment of array 100 of wells. FIG. 2A is a cross-sectional view. Schematic of another exemplary embodiment of array 100 of wells. FIG. 2B is a top view of an exemplary embodiment of an array of wells cut along line BB of FIG. 2A. Schematic of another exemplary embodiment of array 100 of wells. FIG. 3A is a cross-sectional view. Schematic of another exemplary embodiment of array 100 of wells. FIG. 3B is a top view of an exemplary embodiment of an array of wells cut along line BB of FIG. 3A. Schematic of another exemplary embodiment of array 100 of wells. FIG. 3C is a diagram of an array of wells in the shape of a sinusoidal or paraboloid. Schematic of another exemplary embodiment of array 100 of wells. FIG. 3D is a side view of an array of wells containing spheroids in an embodiment. Schematic showing another exemplary embodiment of an array of spheroid-containing cell culture compartments or wells in a wavy embodiment. FIG. 4A is a top view of a wavy embodiment of a substrate having an array of wells. Schematic showing another exemplary embodiment of an array of spheroid-containing cell culture compartments or wells in a wavy embodiment. FIG. 4B is a partial cut-out view of the same embodiment. Schematic showing another exemplary embodiment of an array of spheroid-containing cell culture compartments or wells in a wavy embodiment. FIG. 4C is a top view of a wavy embodiment of a substrate having an array of wells. Schematic showing another exemplary embodiment of an array of spheroid-containing cell culture compartments or wells in a wavy embodiment. 4D is a partial cut-out view of the same embodiment. Schematic of an additional exemplary embodiment in which a series of pillars define the wells. FIG. 5A is an upper view. Schematic of an additional exemplary embodiment in which a series of pillars define the wells. FIG. 5B is an upper view. Schematic of an additional exemplary embodiment in which a series of pillars define the wells. FIG. 5C is a partially cut-out perspective view of an exemplary embodiment. Schematic of an additional exemplary embodiment in which a series of pillars define the wells. FIG. 5D is a partially cut-out perspective view of an exemplary embodiment. Schematic of an additional exemplary embodiment in which a series of pillars define the wells. FIG. 5E is a partially cut-out perspective view of an exemplary embodiment. An exemplary and non-limiting example of the cross-sectional shape of a ridge protruding from a side wall An exemplary and non-limiting example of the cross-sectional shape of a ridge protruding from a side wall An exemplary and non-limiting example of the cross-sectional shape of a ridge protruding from a side wall An exemplary and non-limiting example of the cross-sectional shape of a ridge protruding from a side wall An exemplary and non-limiting example of the cross-sectional shape of a ridge protruding from a side wall An exemplary and non-limiting example of the cross-sectional shape of a fissure in a side wall An exemplary and non-limiting example of the cross-sectional shape of a fissure in a side wall An exemplary and non-limiting example of the cross-sectional shape of a fissure in a side wall An exemplary and non-limiting example of the cross-sectional shape of a fissure in a side wall An exemplary and non-limiting example of the cross-sectional shape of a fissure in a side wall Culture flask with micropatterned bottom surface in an array of microwells Enlarged view of the micro-patterned substrate with an array of microwells forming the bottom surface of the flask shown in FIG. HT29 cell spheroids inside the microwells of the micropatterned T25 spheroid forming flask shown in FIG. Spheroids taken from a micropatterned T25 spheroid formation flask Micrographs of spheroids or 3D aggregates of human ESC cells formed on the NUNCLON SPHERA ™ low-binding surface commercially available from Nunc / Thermo Fisher. Micrographs of spheroids or 3D aggregates of mouse ESC cells formed on a low-binding surface of NUNCLON SPHERA ™ marketed by Nunc / Thermo Fisher. The figure which shows the manufacturing method of the base material which has a multi-well array according to an embodiment. Graph showing viable cell counts measured after proliferation of cells in 6-well plates with substrates with arrays of microwells (as described in Example 1) in microwells with different bottom thicknesses. Graph comparing viable cell counts for substrates with an array of microwells against a flat surface Graph comparing cell proliferation rates for substrates with an array of microwells against a flat surface Graph showing the total titer of proteins excreted from MH677 cells cultured on a substrate having an array of microwells against a flat surface. Image of a structured surface embodiment Photograph of cells grown in wells of structured surface embodiments A side view showing an embodiment of a cell culture apparatus including a porous membrane support. A side view showing an additional embodiment of a cell culture apparatus including a porous membrane support. Side view showing an additional embodiment of a cell culture apparatus comprising a porous membrane support showing co-culture of cells. Schematic perspective view of an embodiment of a cell culture apparatus Schematic cross-sectional view of an embodiment of a cell culture apparatus Schematic cross-sectional view of an embodiment of a cell culture apparatus Schematic bottom view of an embodiment of a tray that can be used to form a portion of the apparatus shown in any of FIGS. 21-23. Schematic perspective view of the tray embodiment shown in FIG. 24A. Schematic side view of the embodiment of the cell culture apparatus Perspective view of an embodiment of a cell culture apparatus having a well Schematic cross-sectional view of an embodiment of a plurality of wells Schematic cross-sectional view of an embodiment of a plurality of wells Enlarged schematic cross-section of a plurality of well embodiments Schematic perspective view of an embodiment of a cell culture apparatus having plates and wells Schematic perspective view of an embodiment of a cell culture apparatus having plates and wells Schematic perspective view of an embodiment of an insert with a cell culture device and a mesh Schematic perspective view of an embodiment of an apparatus having an inlet and an outlet Graph showing protein production per ^{cm 2} in 96-well spheroid microwell plates in CHO5 / 9 alpha cells BHK-21 pc. Graph showing protein production per ^{cm 2} in 96-well spheroid microwell plates in DNA3-1HC cells

Various embodiments of the present disclosure will be described in detail with reference to the drawings. References to various embodiments do not limit the scope of the invention. In addition, the examples described herein are not limiting, but merely describe some of the many possible embodiments of the invention described in the claims. Similar numbers used in the drawings indicate similar elements, processes, and the like. However, it will be understood that the use of a number to refer to an element in a given drawing is not intended to limit that element in another figure with the same number. In addition, the use of different numbers to refer to elements is not intended to indicate that the differently numbered elements cannot be the same or similar to other numbered elements.

All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are intended to facilitate the understanding of certain terms frequently used herein and are not intended to limit the scope of this disclosure.

In certain embodiments, the devices described herein and the methods of making and using such devices provide one or more advantageous features or embodiments, including, for example, those discussed below. The features or aspects listed in the claims are generally applicable to all aspects of the invention. Any one or more described features or aspects in any one claim may be combined with, or in those features or aspects, any other described feature or aspect in any other claim. Can be replaced.

Terms such as "Include, include" mean that they include, but are not limited to, being inclusive and not exclusive.

Values such as, for example, the amount, concentration, volume, treatment temperature, treatment time, yield, flow rate, pressure, viscosity, etc. of the components in the composition and their ranges or components used in the description of the embodiments of the present disclosure. The "about" that modifies the values such as the dimensions of and the range thereof is, for example, used in the preparation of materials, compositions, composites, concentrates, components, manufactured articles, or use formulations. Through typical measurement and handling procedures; through inadvertent errors in these procedures; through differences in the purity of the starting materials or raw materials used to carry out the manufacture, source, or method; etc. Point to that. The term "about" also results from different amounts due to deterioration of the composition or formulation or mixture having a particular initial concentration, and from the mixing or processing of a composition or formulation or mixture having a particular initial concentration. And also include different amounts.

"Optional" or "as needed" occurs / exists or does not occur, even though the steps, features, conditions, properties, or structures described thereafter are within the stated range. / Means that it does not exist.

The terms "favorable" and "preferably" refer to embodiments of the present disclosure that may provide a particular benefit under certain circumstances. However, under the same or other circumstances, other embodiments may be preferred. Furthermore, the description of one or more preferred embodiments does not imply that the other embodiments are not useful and is intended to exclude the other embodiments from the scope of the art according to the invention. Absent.

The devices described herein, methods of making them, and methods of using them may include, in addition to the components or steps described herein, other components or steps not described herein.

As used herein and in the appended claims, the term "or" is generally used to include "and / or" unless otherwise stated. The term "and / or" means one or all of the listed elements, or any combination of two or more of the listed elements.

As used herein, "have, has, having", "includes, includes, including, compute, complements, complementing" and the like are open ended inclusive. Used in a sense, it generally means "include, but not limited to; includes, but not limited to; including, but not limited to".

The range may be expressed herein as from "about" one particular value and / or "about" another particular value. When such a range is represented, examples include from that one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent "about", it is understood that the particular value forms another aspect. It will also be further understood that each endpoint of the range represents both meanings in relation to the other endpoints and independently of the other endpoints.

As used herein, nouns refer to at least one or more objects, unless otherwise stated.

Abbreviations familiar to those skilled in the art can be used (eg, "h" or "hrs" for time, "g" or "gm" for grams, "mL" for milliliters, and room temperature. "Rt", "nm" for nanometers, etc.).

Specific and preferred values for components, ingredients, additives, dimensions, conditions, and similar embodiments, as well as their range, are for illustration purposes only; they are other unless otherwise stated. Do not exclude defined values or other values within the defined range. The devices and methods of the present disclosure are any value described herein, including explicit or implied intermediate values and ranges between them, or any value thereof, a particular value, a particular value, and a preferred value. Includes any combination of.

Similarly, in the present specification, the description of the numerical range by the end points includes all the numbers included in the range (for example, 1 to 5 are 1, 1.5, 2, 2.75, 3, 3. 80, 4, 5, etc.). If the range of values is "greater than" a particular value, "less than" a particular value, etc., the number is included within that range.

The directions referred to herein, such as "top," "bottom," "left," "right," "up," "down," "top," "bottom," and other directions and orientations, are , The drawings are described herein for the purpose of clarity and are not intended to limit the actual device or system, or the use of that device or system. Many devices, articles, or systems described herein can be used in many directions and orientations. As used herein with respect to a cell culture device, the directional descriptor often refers to the direction in which the device is oriented for the purpose of culturing cells within the device.

Unless otherwise stated, no method described herein is intended to be construed as requiring the steps to be performed in a particular order. Therefore, it is otherwise stated in the claims or description that the claims of the method do not actually describe the order in which the steps should be followed, or that the steps should be limited to a particular order. If not, it is never intended that any particular order be inferred. Any one or more described features or aspects in any one claim may be combined with, or in those features or aspects, any other described feature or aspect in any other claim. Can be replaced.

It should also be noted that the description herein refers to a component that has been "configured" or "adapted" to function in a particular manner. In this regard, when such a description is a structural description as opposed to a description of the intended use, such a component embodies a particular property or function in a particular embodiment. As such, it is "configured" or "adapted". More specifically, in the present specification, the reference to an embodiment in which a component is "constituent" or "adapted" means the physical condition in which the component exists, and thus the component of the component. It should be regarded as a clear description of structural features.

Various features, elements, or steps of a particular embodiment may be disclosed using the transition phrase "contains", but using the transition phrase "consists of" or "consists of essentially". It should be understood that alternative embodiments, including those that may be described, are implied. Thus, for example, alternative embodiments impregnated in a cell culture apparatus containing a structure defining a plurality of wells include an embodiment having a structure in which the cell culture apparatus defines a plurality of wells, and a cell culture apparatus. Includes embodiments that are essentially composed of structures that define multiple wells.

In various embodiments, the present disclosure describes a device, such as a cell culture device, that comprises a substrate that defines wells (eg, microwells). The well comprises one side wall (or multiple side walls), a well bottom (or bottom), and an open top (eg, top opening). In embodiments, wells are configured to include aqueous liquid compositions such as those used in cell cultures or cell assays. For example, the aqueous liquid composition can include cell culture media, buffers or other solutions or mixtures used in cell assays.

The embodiments described herein find applications using any device, eg, with small (eg, microscale) wells or other containers, or chambers configured to contain a liquid. In certain embodiments, the wells of the devices described herein find use in culturing cells. More specifically, the device and the microwells within it find use in 3D cell culture of cell aggregates or spheroids.

There are several different shapes used to culture cells as aggregates. In some embodiments, the cell aggregate is a cell mass, embryoid body, or spheroid. A common geometry for forming cell aggregates is the hemisphere found on the rounded well-bottomed microplate. In some embodiments, a non-adhesive surface is used to prevent cells from adhering to the surface. The non-adhesive material may be applied after the well or chamber (eg, in the microplate) has been manufactured, or the well or chamber material may have innate non-adhesive properties.

FIG. 1 is a schematic representation of an exemplary embodiment of an array of wells 100 showing individual wells 115. In the embodiment shown in FIG. 1, the well 115 has a mouth 110. The mouth 110 is the top of the well adjacent to the top opening 111 of the well 115, which results in a larger open area before the well shrinks and cells settle to form spheroids and form the bottom of the well. Area of. In embodiments, the mouth 110 can be conical (wider at the top of the mouth than at the bottom of the mouth) and ring-shaped (as shown in FIGS. 1A and 2A where the wells are circular). In additional embodiments, for example, if the well has a circular opening but is in the shape of a paraboloid, as shown in FIG. 3A, the mouth 110 can be a paraboloid, or FIG. 27. And as shown in FIG. 28, the mouth can extend into each well 115. In the embodiment, there is no mouth. The presence of the mouth structure can provide two functions. First, the mouth widens the opening of the well, allowing liquid to be introduced into the opening of the well and flow down to the bottom of the well. This promotes cell aggregation at the bottom of the well and promotes spheroid formation in culture. In addition, the mouth provides a geometric feature that can create a transition from the annular inner surface of the mouth to the inner surface of the well, thereby preventing air from being trapped in the well. The presence of a 90 degree angle between the top of the well and the side wall of the well may provide a place for the formation of bubbles. The mouth provides a transition between the top and side walls of the well that is not at a 90 degree angle, thereby reducing the formation of air bubbles within the well having the mouth structure.

The dimensions of the wells for use in aggregate cell culture methods can be approximately micrometers to millimeters (eg, 100 μm to 50 mm). Devices with wells for cell culture are commercially available from many different manufacturers (eg, Corning, Nunc, Greener, etc.). "Microwells" generally have dimensions of about micrometers (eg, ≤1 mm, ≤500 μm, ≤400 μm, ≤200 μm) or a few millimeters (eg, ≤10 mm, ≤5 mm, ≤3 mm, etc.) and aggregates. Wells that can also be used for cell proliferation. In some embodiments, microwells provide containment for 3D cell culture. In a suitable embodiment herein, the term "well" explains or suggests that the context is not (eg, if the well is described as having a well bottom with multiple microwells). ) Except for the use of microwells. Wells intended to be non-microwell dimensions may be referred to as "macro wells" or simply "wells." In some embodiments, the height of the well or the depth of the well (eg, from the top opening to the bottom of the well) is greater than or equal to 100% (eg, 100%, 110%, 120%) of the diameter of the well at the top opening. 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, or theirs. Any range between). In some embodiments, the depth of the well (eg, from the top opening to the bottom of the well) is greater than or equal to 100% (eg, 100%, 110%) of the diameter of the well at the midpoint between the top opening and the bottom of the well. , 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400 %, Or any range between them).

One of the most commonly used microwell products is a commercially available "commercial" that presents an inverted pyramid-shaped geometry with a diameter of 400 or 800 micrometers, arranged at the bottom of a standard format microplate well. "Aggrewell" plate (commercially available from Stem Cell technologies). Another geometry for growing cells as aggregates is the "Elprasia" microplate (Kuraray) using "microspace cell culture"; these plates allow cells to aggregate, standard. It has square microwells 200 micrometers in diameter arranged at the bottom of the format microplate wells. Various parameters, dimensions, and methods for making microwells for culturing cells as aggregates are understood in the art (the whole of them is incorporated herein by reference). U.S. Patent Application Publication No. 2004/0125266; U.S. Patent Application Publication No. 2012/0064627; U.S. Patent Application Publication No. 2014/02277784; International Publication No. 2008/106771; International Publication No. 2014/165273). U.S. Pat. No. 6,348,999 describes fine concavo-convex elements and how they are constructed without reference to the purpose of these structures other than as polymer lens arrays. U.S. Pat. Nos. 5,151,366, 5,272,084, and 6,306,646 are described in order to increase the surface area for cell adhesion on the substrate. Containers with various types of fine concavo-convex patterns and methods of making culture patterns have been described, but the patterns themselves will not promote the formation of cell aggregates. Other devices, compositions, reagents and methods are incorporated herein by reference, eg, in their entirety, in the art, U.S. Patent Application Publication No. 2014/0322806; U.S. Pat. No. 8,906. , 685; Haycock. Methods Mol Biol. 2011; 695: 1-15; US Patent Application Publication No. 2014/0221225; International Publication No. 2014/165273; US Patent Application Publication No. 2009/0018033 It is described in the book.

The geometry of some commercially available wells promotes the formation of cell aggregates, but not necessarily "confinement". If aggregated cells are not trapped, they usually grow as large as their environment allows. Cell aggregates> 150 to 400 micrometers in diameter (depending on the cell type) can form a necrotic core. Necrosis results from, for example, that the cell mass is too large to limit the diffusion of nutrients into the center of the aggregate and the discharge of metabolic waste out of the center of the aggregate. In some embodiments, it closely resembles the desired maximum diameter dimension of cell aggregates to cause confinement (eg, within 50%, within 40%, within 30%, within 20%, 15). Within%, within 10%, within 5%, within 2%, within 1%, or within the appropriate range within them, but at least 1.5 to 2 times the diameter (eg, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.52.6, 2.6, 2.8, Microwell geometry is used, which is 3.0, 3.5, 4.0, or within the appropriate range within them. In some embodiments, the geometry of the confinement well also allows the exchange of liquid culture medium through perfusion or manual pipetting without lifting the cell spheroids out of the confinement well.

Existing devices (eg, microplates in microwell format or other containers with microwells) exhibit design flaws that adversely affect the use of such devices for the formation of 3D cell aggregate structures. Although handling of microwell-formatted microplates is fairly straightforward, problems often arise if air is not removed from the microwells during the introduction of a liquid (eg, medium). Air uptake is a common problem with microplate wells having a geometry of 384-well format size, especially when the wells are circular. The surface tension of the aqueous liquid is so strong that the droplets remain spherical. Spherical droplets can close circular holes of similar size (eg, well cross-sectional shape). The presence of air in a well adversely affects and / or suppresses cell culture in that well.

In order to grow spheroids in a cell culture vessel at high density on a substrate having an array of spheroid formation wells with a non-adhesive surface, a culture surface that balances many variables is required. For example, the maximum spheroid density achievable, the ability to maintain the position of spheroids during fluid exchange operations while allowing them to be removed if desired, and the spheroid formation wells when the container is filled. A balanced design, such as avoiding the uptake of air inside, is highly desirable to avoid problems when handling such containers. Embodiments herein provide a well geometry that maintains confinement dimensions, aids in the elimination of air in the microwells, and allows the inflow of liquid into the microwells. Addresses the problem of air uptake in traditional wells. For example, a square top well, which has a rounded well bottom geometry, allows air to travel up the corners of the well around the aqueous droplets, thus capturing air when adding liquid. Very unlikely. In some embodiments, air is introduced into the well by including various structures extending from the well opening to the well bottom (eg, pillars, corrugations, corners, ridges, crevices, etc.). It provides a route for escape. In addition to the features described herein, some embodiments also include geometric shapes, materials, etc. described and / or understood in the art.

One of the commonly used design features to avoid the problem of air uptake in high density spheroid growth substrates is the avoidance of stepped changes in sharp corners or the geometry of the substrate. In particular, avoiding those running at right angles to the flow path of the liquid across the surface. For example, the AggreWell plate has a wall that slopes at an angle close to 90 degrees. This facilitates drainage from the surface as the container is filled, leaving air-filled wells.

A surface geometry that addresses the problem of air uptake during liquid introduction, which maintains well features that facilitate the growth and maintenance of high density of individual spheroids (eg, rounded well bottoms). Provided on the specification.

In some embodiments, well-shaped transitions are used to alleviate the problem of escaping air as the liquid is introduced into the wells. For example, in some embodiments, a well bottom (or well bottom portion) of a circular cross section is used for spheroid formation. However, the circular cross section can be particularly problematic for allowing air to escape without the formation of pockets. To alleviate this problem, wells are formed with a circular well bottom cross section and a non-circular (eg, triangular, square, rectangular, pentagonal, hexagonal, etc.) top opening. In such an embodiment, the sidewalls transition from a non-circular (eg, polygonal) top opening to a circular well bottom. In some embodiments, the transition is an interfering, jagged, or horizontal side wall feature that can cause a "hanging up" of bubbles escaping from the well as the liquid is introduced into the well. Gradually migrate so as not to introduce. In some embodiments, the corners of the side walls created by the non-circular (eg, polygonal) shape of the transitioning wall and top opening provide a path for liquid inflow and / or air escape. To do.

In some embodiments, the geometry of the well has a capillary structure (eg, mouth, ridge, crevice, rounded) within the well wall to facilitate air bleeding as the liquid is introduced into the well. Or including the upper opening of the paraboloid). FIG. 1B is an upper view taken along line BB of FIG. 1A showing the ridge 170. As shown in FIG. 1B, the ridge is a bump or protrusion from the mouth 110 or side wall 113 of the well. In an embodiment, the ridge extends the length of the microwell from the top opening 111 to the well bottom 116. In additional embodiments, the ridge extends from the top 111 of the mouth to the bottom 112 of the mouth. The acute angles formed on either side of the ridge 170 create capillary forces in the aqueous fluid, resulting in the inflow of the fluid into the microwells without air pools. FIG. 2B is a top view of the well array 100 showing a cross section in FIG. 2A. FIG. 2B shows the crevice 270. As shown in FIG. 2B, the fissure is an indentation on the side wall 113 of the well 115. The acute angles formed on either side of the crevice 270 create a capillary force that allows the aqueous fluid to flow into the microwells.

3A and 3B are schematic views of another exemplary embodiment of the array 100 of wells. FIG. 3A is a cross-sectional view. FIG. 3B is a top view of an exemplary embodiment of an array of wells cut along line BB of FIG. 3A. 3A and 3B show that each well 115 can have more than one ridge 170 or crevice 270, and that the ridge 170 or crevice 270 can be arranged in an array within the well 115. As shown in FIGS. 3A and 3B, radial distribution of ridges and / or fissures is assumed in embodiments. The number of capillary structures is not limited to one per microwell. In some embodiments, the higher the number of capillaries, the slower the rate of fluid inflow into the microwells.

3A-D show multiple (eg, 2,3,4,5,6,7,8,9,10,12,16,20,24,28,32, or theirs) in a single well. Demonstrates to include vertically oriented capillary structures (in any range of). The features may be spaced at regular intervals (as shown in FIG. 3), may be spaced at irregular intervals, may be grouped / raised, and the like. In some embodiments, the capillary structure extends from the top opening of the well to the bottom of the well. If there are multiple capillary structures within a single well, the features may be of different types (eg, ridge lines and / or crevices) and of different shapes (eg, square, circular, etc.). May include.

FIG. 3C shows that the array 100 of wells can have a sinusoidal or paraboloidal shape. This shape creates a rounded top edge or well edge, which, in embodiments, reduces air uptake at sharp corners or 90 degree angles at the top of the well. This sinusoidal or parabolic well shape, or rounded upper well edge, is also a capillary structure. As shown in FIG. 3C, wells 115, upper diameter D _top, at the midpoint of the height between the bottom 116 of the upper and the well height H, and the well from the bottom 116 of the well to the top of the well It has an upper opening with a well diameter D _middle.

FIG. 3D is a schematic view of the array of wells described in FIGS. 1-3 and embodiments. FIG. 3D shows a plurality of microwells 115 arranged in an array within the substrate 1114. A plurality of spheroids 500 present in the plurality of microwells 115 are also shown in FIG. 3D.

4A and 4B are schematics showing another exemplary embodiment of a spheroid-containing cell culture compartment, or array of wells. FIG. 4A is a top view of the wave-shaped embodiment of the array of wells, and FIG. 4B is a partial cut-out view of the same embodiment. In the embodiments shown in FIGS. 4A and 4B, the wells are not isolated, allowing the flow of liquid between the wells. As shown in FIGS. 4A-B, exemplary embodiments are shown in which the wavy side walls 403 are aligned to form microwells 401 in the gaps between the wavy side walls 403. There is. The wavy sidewalls shown in FIGS. 4A and 4B are surrounded by a frame 402, which are separated from each other in the cycle created by the rows of microwells 401 and then approach each other (eg, with or without contact). ) Has an area. In some embodiments, the indentations in the microwells are located at the base of each segment, defined by a wall farther away (eg, to accommodate the Spheroid 500). 4C and 4D are additional schematics of a wavy microwell array embodiment. FIG. 4C is an upper view of the embodiment, and FIG. 4D is a perspective view of the embodiment. As shown in FIG. 4D, in the embodiment, there is no frame. 4C and 4D show a wavy or wavy side wall 403 that forms a space 401 with a geometry suitable for inducing spheroid formation. The spheroids 500 present in the wells are also shown in FIGS. 4C and 4D. When the liquid is introduced into the embodiments shown in FIGS. 4A and 4B, the excluded air can move out of the local area through the area where the walls approach each other. The movement of fluids and air in and out of the wider well area is facilitated by the narrows to avoid air uptake. These waveforms also promote spheroid formation by providing a reduced proliferative region. That is, the waveform is a capillary structure and a geometric shape that induces spheroids.

5A and 5B show a schematic top view of an exemplary embodiment of an array of wells 100 in which a series of pillars define the wells. 5C-5E are perspective views of an embodiment of an array of microwells showing pillars 501 arranged in an array to form wells 515. In embodiments, the top 510 of pillar 501 can be flat (shown in FIG. 5A). In this embodiment, the convex wall created by the pillar 501 creates a very sharp angle or discontinuity at the junction between the pillar forming sidewalls. FIG. 5B shows a microwell recess surrounded by pillars to create a confinement geometry suitable for inducing the formation of spheroid 500 in culture. Air escapes and fluid enters through the space 505 between the pillars 501. Pillar 501 has an upper 510. In an embodiment, the pillar top 510 may be rounded, as shown in FIGS. 5B, 5D and 5E, which will give rise to wells having the parabolic or sinusoidal shape shown in FIG. 3A. Pillars also promote spheroid formation by providing reduced proliferative regions. That is, the pillars have a capillary structure and a geometric shape that induces spheroids.

For example, ridges, crevices, ridges, dibots, open ring structures of top openings or mouth structures, pillars, discontinuous walls, mouth structures, parabolic or sinusoidal well shapes, rounded well openings, etc. Or these structures, including breaks in the smooth inner surface of the side wall of the well, or any combination of these features, are capillary structures. Capillary structure also provides a route to allow air to escape that can be trapped after the addition of liquid. In some embodiments, discontinuous walls, walls with ridges or crevices, or other features that interrupt the smoothness of the side walls of the wells are provided by providing ventilation areas within the wells during filling. Used to avoid ingestion of air.

In some embodiments, the capillary structure extends along the vertical length of the well wall. In some embodiments, the capillary structure extends to or above the top opening of the well. In other embodiments, the capillary structure extends near the top opening of the well (eg, <0.1 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm from the top opening. 5, 10 μm, or 20 μm (or the range between them). In some embodiments, the capillary structure extends to the bottom of the well. In other embodiments, the capillary structure extends close to the bottom of the well (eg, <0.1 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 10 μm from the bottom of the well. , Or 20 μm (or the range between them)).

In some embodiments, the capillary feature provides the benefit that the liquid entering the well provides a pathway for movement without taking air into the well. In some embodiments, the capillary structure provides a pathway for air exiting the well in response to the introduction of liquid entering the well. The technique is not limited to a specific mechanism of action for preventing the uptake of air, and understanding of the mechanism is not required for the practice of the present invention.

If rapid vessel filling or another fluid introduction event leads to air uptake, the capillary feature is trapped air, despite the well geometry configured to prevent such uptake. Allows the movement of liquid underneath, thereby expelling air from the wells. For example, in the wavy embodiment shown in FIG. 4, liquids and air can flow through an open well structure from one well region to the next through the narrow portion of the array. Alternatively, in the pillar embodiment shown in FIG. 5, liquid or trapped air can exit the well through the space between the pillars. In some embodiments, the downward force of the liquid through the capillary structure serves to separate the air from the well wall and surround the air with the liquid, thereby raising the air pool as bubbles out of the well.

A variety of different vertically oriented structures find use as capillary features. For example, in certain embodiments, the feature is a raised ridge (eg, as shown in FIGS. 1A and 1B), or a hidden groove or fissure (eg, as shown in FIGS. 2A and B). .. Ridge lines and crevices for which use is found in the embodiments herein are not limited to the physical geometry shown. In some embodiments, the features extend vertically along the side walls of the well without moving horizontally. In most cases, the horizontal structural features facilitate the uptake of air in the wells in a manner similar to the steep angles used for existing well geometry. Suitable cross-sectional shapes of the ridge are shown in FIG. 6, which are rounded (FIG. 6A), angular (FIG. 6B), needle-like (FIG. 6C), and semi-hexagonal (FIGS. 6D and 6E) shapes. And so on. Suitable cross-sectional shapes of the crevice are shown in FIG. 7, which are rounded (FIG. 7A), angular (FIG. 7B), needle-like (FIG. 7C), and semi-hexagonal (FIGS. 7D and 7E) shapes. And so on. Ridges and / or crevices can be of any suitable cross-sectional size (eg 0.1, 0.2, 0.5, 1, 2, 5, 10). , 15, 20, or any suitable range between them) has a width, length, etc.). To optimize the shape and dimensions of ridges and / or crevices to facilitate fluid introduction and air drainage without forming air pools below the liquid surface, and / or to remove trapped air pools. To facilitate, engineering and microfluidic principles may be used in combination with embodiments herein.

In some embodiments, the movement of liquid into the wells and the movement of air out of the wells is through the geometry of the discontinuous side walls. The geometry of the discontinuous wells takes the form of discontinuities in the side walls of the wells. Examples of discontinuous side wall shapes are as shown in the "wavy wall" or "wavy" geometry of FIG. 4 and the "pin wall" or "pillar wall" geometry of FIG. It is a thing. These shapes are merely illustrations, and the orientation of other sidewalls that introduce gaps or other discontinuities in a portion (eg, top) of the sidewalls may find use in the embodiments herein. In these geometries, breaks in the wall allow air to move rapidly out of the wells as the fluid enters the vessel, due to its low stickiness. In some embodiments, the discontinuous geometry maintains avoidance of sharp changes in the wall features of the substrate (eg, avoidance of features that create horizontal obstacles). In some embodiments, the discontinuous side wall geometry is used in conjunction with well-shaped transitions and / or capillary wall structures to aid in the release of bubbles and / or the release of air during liquid introduction. ..

In some embodiments, the one or more wells have a concave curved surface, such as a conical surface with a hemispherical or round bottom, and similar surface shapes or combinations thereof. Wells and well bottoms are finally terminated, terminated, or bottomed on rounded surfaces or curved surfaces, such as recesses or wells, and similar concave truncated cone undulating surfaces, or combinations thereof. out) can be done. Other shapes and configurations of wells that facilitate spheroids are incorporated herein by reference in their entirety, unless inconsistent with the present disclosure. / 087,906. In embodiments, the bottom of the well is flat or tapered. The well bottom may have any other suitable shape or size. For example, in embodiments, the bottom of the well has a rounded surface or curved surface, or the bottom of the well promotes the formation of spheroids by providing a reduced growth area, recesses, recesses, and similar recesses. It can have structures such as truncated cone-shaped undulating surfaces, multiple indentations or nib regions, or a combination thereof. That is, a rounded or curved or recessed well bottom or nib region or corrugation or pillar is a spheroid-inducing geometry.

An exemplary well geometry and dimensions are shown, for example, in FIGS. 1 and 2. In some embodiments, the wells 100 described herein are in the range between any two of the following values (eg, 200-1000 μm, 200-750 μm, 300-750 μm, 400-600 μm). Etc.), eg, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, or 2000 micrometers, etc. It has a well bottom diameter of 130/230 in the range of 100 micrometers to about 2000 micrometers. Such diameter dimensions control the dimensions of the spheroids that proliferate in them so that the cells inside the spheroids remain healthy. That is, these dimensions promote the formation of spheroids by providing a reduced growth area. That is, these dimensions are geometric shapes that induce spheroids.

In some embodiments, the wells 100/200 described herein include a range between any two of the following values, eg, 100, 150, 200, 250, 300, 350. , 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, or 2000 micrometers, the cross-sectional dimensions of the upper opening in the range of about 100 micrometers to about 2000 micrometers 120. Has / 220 (eg, diameter or width). In some embodiments, the well 100 comprises a range between any two of the following values, eg, 100, 200, 300, 400, 500, 600, 700, 800, 900, or. It has a depth of 160/260 from the top opening to the bottom of the well in the range of about 500 micrometers to about 1500 micrometers, such as 1000 micrometers. In some embodiments, the well 100/200 includes a range between any two of the following values, eg, 50, 60, 70, 80, 90, 100, 200, 300, 400. , Or having an upper part with a depth of 140/240 in the range of about 50 micrometers to about 500 micrometers, such as 500 micrometers. In some embodiments, the well 100 includes a range between any two of the following values, eg, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600. , 700, 800, 900, 1000, 1200, or 1400 micrometers, with a lower part having a depth of 150/250 in the range of about 100 micrometers to about 1400 micrometers. Of course, other suitable dimensions can also be used.

In some embodiments, the structure and properties of the transition well, as well as one or more design elements and / or physical features configured to allow air to escape upon introduction of the liquid into the well. In addition, the devices and wells described herein (eg, microwells) may have additional features, eg, to provide specialized functionality for 3D culture of cells within microwells. The following paragraph addresses such features that may be used in combination with the embodiments described above.

In some embodiments, all or part of the side walls and / or bottom of the well is gas permeable. In some embodiments, gas permeability allows the transfer of oxygen and other gases dissolved in the liquid or medium contained within the wells into the wells. Permeable sidewalls, well bottoms, or parts of them do not form air pools or bubbles in the well liquid.

In some embodiments, a cell culture device is provided that has a structured surface that defines multiple gas permeable wells. In some embodiments, the wells may include an outer surface that defines the outer surface of the device. In some embodiments, the well may include an external or non-cultured surface communicating with the outside of the device. In some embodiments, among others, a cell culture apparatus having a plurality of stacked cell culture compartments, each having a structured surface defining a plurality of gas permeable wells, is provided herein. Will be done. In some embodiments, the wells in various embodiments gas communicate with the outside of the device, such as indirectly through vents or through conduit spaces, or directly through external walls.

In some embodiments, the wells are configured such that cells cultured in the wells form spheroids. For example, in some embodiments, the wells are non-adhesive to the cells so that the cells within the wells associate with each other (eg, form spheroids). The spheroid extends to the dimensional tolerance imposed by the well geometry. In some embodiments, the wells are coated with an ultra-low binding material to make the wells non-adhesive to cells.

The formation of three-dimensional (3D) cell aggregates, such as spheroids, increases the density of cells growing in a cell culture device, as opposed to two-dimensional cell culture, in which cells form a monolayer on the surface. This can also increase the nutritional requirements of the cells cultured in the device. Since the exchange of metabolic gas can occur through the gas permeable wells in which the cells are cultured, the medium volume in the cell culture apparatus is essentially that the exchange of metabolic gas causes diffusion through the cell culture medium. It can be larger than is possible with restricted equipment. Therefore, the height of the cell culture medium used in the cell culture apparatus described herein can be increased, and therefore the volume can also be increased.

In some embodiments, the cells are cultured in the wells of the apparatus described herein, where the cell culture medium is at a height of 2 mm or more above the cells. In some embodiments, the cell culture medium is at a height of 5 mm or more above the cells. Exchange of metabolic gas, such as when the substrate on which the cells are cultured, or its surface, or near it, is opaque or relatively opaque to the metabolic gas (eg, compared to cell culture medium). A maximum height of 2 mm to 5 mm of cell culture medium is generally considered to be the upper limit of the height of the medium if is essentially restricted to exchange through the medium.

For the purpose of efficient metabolic gas exchange, in some embodiments, the cell culture medium in the apparatus is arbitrary on the cells, such as 2 mm or more above the cells, 5 mm or more above the cells, or 10 mm or more above the cells. When at the height of, cells are maintained in the culture of wells of the devices described herein. However, those skilled in the art will appreciate that as the height of the cell culture medium in the device increases above the cells, so does the hydrostatic pressure exerted on the cells. Therefore, there may be practical restrictions on the height of the cell culture medium above the cells. In some embodiments, the height of the cell culture medium above the cells cultured in the wells of the articles described herein comprises a range between any two of the following, eg, 5, 6, It is in the range of 7, 8, 9, 10, 15 or 20 mm, for example, 5 mm to 20 mm such as 5 mm to 15 mm, 6 mm to 15 mm, 5 mm to 10 mm or 6 mm to 10 mm.

In certain embodiments, the cell culture substrate or layer having a non-cultured surface that is gas communication with the outside of the device will have a structured surface that defines the gas permeable wells described herein. Can be adapted to. Examples of such a cell culture apparatus include a T-flask, a TRIPLE-FLASK cell culture vessel (Nunc., Inc.), a HYPERFLASK cell culture vessel (Corning, Inc.), and a CELLSTACK culture chamber (Corning, Inc.). ), CELLCUBE module (Corning, Inc.), CELL FACTORY incubator (Nunc, Intl.), And, to the extent consistent with the disclosures presented herein, by reference in their entirety. International Publication No. 2007/015770, U.S. Patent Application Publication No. 2014/0315296, U.S. Pat. No. 8,846,399, U.S. Pat. No. 8,178,345, and U.S. Patent No. 7 incorporated herein. , 745, 209, the cell culture articles described in each specification.

In some embodiments, gas-permeable / liquid-impermeable materials are used to construct cell culture devices herein, or parts thereof (eg, wells, microstructures, etc.). Any suitable gas permeable / liquid permeable material, such as polystyrene, polycarbonate, ethylene vinyl acetate, polysulfone, polymethylpentene (PMP), polytetrafluoroethylene (PTFE) or compatible fluoropolymers, silicones. Rubber or copolymer, poly (styrene-butadiene-styrene), polyolefin such as polyethylene or polypropylene, or a combination of these materials can be used. The substrate can be formed from any suitable material that has suitable gas permeability in at least a portion of the wells. Examples of suitable substrates include polydimethylsiloxane (PDMS), (poly) 4-methylpentene (PMP), polyethylene (PE), and polystyrene (PS). PDMS can have a high degree of gas permeability and can achieve sufficient gas permeability at thicknesses up to 40 mm. PMP can achieve sufficient gas permeability at thicknesses up to about 01 mm. In some embodiments, the PMP has a thickness in the range of about 0.02-1 mm. Although thinner substrates may not have sufficient structural integrity, PE or PS can achieve sufficient gas permeability at thicknesses up to 0.2 mm. To compensate for poor structural integrity, opening frames, standoffs, etc. can be used to support the substrate from the bottom. In embodiments, well thicknesses include a range between any two of the following: 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, It can be 20 or 40 mm. In embodiments, the wells have an oxygen permeation rate through a gas permeable polymer material of ^{2000 cm 3} / m ^{2 / day or greater.} In some embodiments, the wells have gas permeability through a substrate of ^{3000 cm 3} / m ^{2 / day or more.} In some embodiments, the wells have gas permeability through a substrate of ^{5000 cm 3} / m ^{2 / day or more.}

Such materials allow for effective gas exchange between the outer and inner compartments, preventing the passage of liquids or contaminants while allowing the ingress of oxygen and other gases.

In some embodiments, the thickness of the well substrate material is adjusted to allow optimized gas exchange. In some embodiments, the well thickness is 10-100 μm (eg, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, and theirs. Any range between). Experiments performed during the development of the embodiments herein have demonstrated that the use of thinner wells produces more viable cells (eg, 17 μm> 30 μm> 57 μm).

FIG. 8 is a diagram showing an embodiment including a substrate having an array of microwells, which is included as the surface of a cell culture apparatus. In FIG. 8, the cell culture device is a flask 800. However, in embodiments, the substrate is a multi-well plate, flask, dish, tube, multi-layer cell culture flask, bioreactor, or any other study intended for cell or spheroid growth. It should be understood that it can form part of any type of cell culture device, including a flask. The substrate with the array of microwells can be a gas permeable material. FIG. 9 shows an enlarged view of the substrate shown in 9, micropatterned with an array of microwells, forming the bottom surface of the flask shown in FIG.

For the embodiments shown in FIGS. 8 and 9, the device illustrated is a flask or enclosure with a cell culture chamber 850. The housing includes a substrate (shown in FIG. 9) having an array of microwells. The housing also has an upper surface 815 and one or more side walls 820 extending from the structured surface 110 to the upper surface 815. In some embodiments, the housing 800 comprises a single surrounding side wall, such as a cylindrical wall. Additional examples of such devices are, for example, the United States Provisional Patent Application No. 62/072 assigned to the assignee of the invention, which is incorporated herein by reference in its entirety, unless inconsistent with the present disclosure. , 015.

The housing is provided with port 860. An opening with a screw lid 861 is shown in FIG. However, in embodiments, the enclosure may have any type of port that allows liquids and cells to move in and out of the enclosure. Port 860 can be on the side wall, top surface 815 or cell culture surface 110. Port 860 may be linked to a tube or other connection for introducing or removing cells and cell culture medium into the cell culture chamber 850.

The housing shown in FIG. 8 shows a fixed side wall 820, but in embodiments, the side wall is flexible or flexible to allow variable volume of cell culture medium that enters the cell culture chamber 850. It can be expandable and foldable. The flexible side wall 820 is extensible as additional cell culture medium is introduced into the cell culture chamber 850 via port 860, and the cell culture medium is extensible through port 860 and cell culture chamber 850. When removed from, the flexible side wall 820 can deflate. In some embodiments, the side wall 820 and top 815 are formed from a bag. In addition, the cell culture chamber 850 can be filled with the volume of cell culture medium up to a fixed volume of the enclosure. In some embodiments (not shown), the entire internal volume or a volume close to it is filled with cell culture medium.

In the embodiment shown in FIG. 8, the volume of the cell culture medium in the cell culture chamber 850 exists _{at the height H m of the medium above the cells.} As described above, the height H _m of the medium on the cells cultured in the housing as described herein, the well can be higher than possible will try height if not gas permeable. In some embodiments, the cell culture chamber 850 is filled to its limit capacity for the purpose of culturing cells in the apparatus.

FIG. 10A shows the spheroid 500 of HT29 cells in the microwell 110 of the micropatterned T25 spheroid forming flask shown in FIG. FIG. 10B shows a spheroid 500 taken from a micropatterned T25 spheroid forming flask, both of which follow Example 2 below. 11A and 11B are microscopes showing a wide range of size distributions of 3D aggregates (human ESC cells of FIG. 11A and mouse ESC of FIG. 11B) formed on a low-binding surface of NUNCLON SPHERA ™ marketed by Nunc. It is a photograph. The NUNCLON SPHERA ™ low-binding surface has a low-cell binding surface treatment, but lacks the geometry disclosed herein that allows the formation of uniform spheroids.

FIG. 12 is a diagram of a method for producing a multi-well array base material according to the embodiment and described in Example 1 below. FIG. 12 shows a thermal embossing / thermoforming method, but other methods of making microwell arrays according to embodiments, including coining, injection molding, embossing and other methods known in the art. The method is also planned.

13, 14A and B, and 15 show viable cell counts (FIGS. 13 and 14A) and cell proliferation rates (FIGS. 14B and 15) for substrates with an array of microwells on a flat surface. ) Is a graph for comparison. The gas permeability of the wells may depend in part on the material of the substrate and the thickness of the substrate along the wells. In the embodiment, the thickness of the side wall and the bottom of the well in the base material having the microarray of the well may be constant and may be relatively thin. Alternatively, in embodiments, the well walls in the array of microwells may be relatively thick near the opening into the well and relatively thin at the bottom of the well. Alternatively, in embodiments, the well walls in the array of microwells may be relatively thin near the opening into the well and relatively thick at the bottom of the well. Depending on the material used and the thickness used, the substrate with the array of microwells can be gas permeable for the purposes of the present disclosure.

FIG. 15 shows a graph showing the total titer of proteins excreted from MH677 cells cultured on a substrate with an array of microwells against a flat surface. These data will be discussed in Example 2 below.

The apparatus shown in FIG. 8 may be a side hard flask or a side soft cell culture flask, but has a surface that defines the outer surface of the cell culture apparatus or is gas-communicated with the outside of the apparatus. It is understood that any other cell culture device with a structured microwell array having can have a substrate having an array of microwells formed from the gas permeable materials described herein. Yeah.

The structured surface of the cell culture apparatus with the array of microwells described herein can define the appropriate number of wells, which can have the appropriate size or shape. Wells determine their volume based on their size and shape. In many embodiments, one or more or all wells are symmetrical and / or rotatable around a longitudinal axis. In some embodiments, the longitudinal axes of one or more or all wells are parallel to each other. Wells can be uniformly or non-uniformly separated. In some embodiments, the wells are evenly spaced. One or more or all wells may have the same size and shape or may have different sizes and shapes.

In some embodiments, the thickness and shape of the substrate around the well is configured to compensate for the reflection of light entering and exiting the inner surface. In some embodiments, the correction is achieved by adjusting the thickness of the substrate material forming the wells. In some embodiments, the thickness of the substrate material closest to the bottom (or bottom) of the well is greater than the thickness of the substrate material closest to the sidewall and / or top opening. In some embodiments, the thickness of the substrate material is progressively reduced from a maximum at the bottom of the well bottom to a minimum at the top opening. For example, the shape and thickness are incorporated herein by reference in their entirety, as long as they are not inconsistent with the present disclosure, in US Provisional Patent Application No. 62/072,019 transferred to the assignee of the invention. It can be described.

For example, a combination of non-adhesive wells, spheroid-inducing well geometry, and gravity can define a confinement volume that limits the growth of cells cultured in the wells, thereby. The result is the formation of spheroids with dimensions defined by the confinement volume.

In some embodiments, the inner surface of well 2115 is non-adhesive to cells. The wells may be formed from a non-adhesive material or may be coated with a non-adhesive material to form a non-adhesive well. Examples of non-adhesive materials include perfluoropolymers, olefins, or similar polymers, or mixtures thereof. Other examples include nonionic hydrogels such as agarose and polyacrylamide, polyols such as polyethers such as polyethylene oxide and polyols such as polyvinyl alcohol, or similar materials, or mixtures thereof. For example, a combination of non-adhesive wells, well geometry, and gravity can induce cells cultured in the wells to self-assemble into spheroids. Some spheroids can maintain differentiated cell function with a greater in vivo-like response compared to cells grown in a monolayer.

In some embodiments, the one or more wells have a concave surface such as a hemispherical surface, a surface geometry such as a conical surface with a round bottom, or a combination thereof. Wells and well bottoms eventually terminate, terminate, or terminate on rounded surfaces or curved surfaces that facilitate spheroids, such as recesses, recesses, and similar truncated cone-shaped undulating surfaces, or combinations thereof. Can bottom out. Other shapes and configurations of wells that promote gas permeable spheroids are incorporated herein by reference in their entirety, unless inconsistent with the present disclosure, the United States of America assigned to the assignee of the invention. It is described in Patent Application No. 14/087,906.

In some embodiments, the bottom of the well is flat or tapered. The well bottom may have any other suitable shape or size.

In some embodiments, the wells 115 described herein include a range between any two of the following values, eg, 200, 250, 300, 350, 400, 450 or 500. It has a diameter dimension w in the range of about 200 micrometers to about 500 micrometers, such as micrometers. Such diametrical dimensions can control the dimensions of the spheroids that proliferate in them so that the cells inside the spheroids remain healthy. In some embodiments, the well 115 comprises a range between any two of the following values, eg, 100, 150, 200, 250, 300, 350, 400, 450 or 500 micrometers. Etc., have a height H in the range of about 100 micrometers to about 500 micrometers. Of course, other suitable dimensions can also be used.

In some embodiments, the structured surface that defines the wells comprises an array of hexagonal close-packed well structures. An image of an embodiment of such a substrate having an array of hexagonal microwells 100 is shown in FIG. 16 showing a substrate having an array of hexagonal wells 1601. FIG. 17 is a schematic diagram showing cells (spheroids) 500 grown in well 1601 of a substrate embodiment having an array of microwells 100 with a hexagonal close-packed well structure. In some embodiments, the cells within each well 1601 form a single spheroid 500, as shown.

FIG. 18 is a side view showing an embodiment of a cell culture apparatus including a porous membrane support. Various embodiments of the cell culture apparatus 2100 incorporating the porous membrane support 2500 are shown in FIGS. 18-20. The porous membrane support 2500 is placed across the enclosure (eg, coupled to one or more side walls 2120) so that the interior of the enclosure is compartmentalized into separate culture chambers 2152 and 2154. Will be done. The first culture chamber 2152 comprises a substrate that forms a structured surface that defines a gas permeable well 2200 that communicates with the outside of the device. The upper part of the chamber 2152 is defined by the porous membrane 2500. The bottom of the second culture chamber 2154 is defined by a porous membrane 2500. Thus, the first population of cells 2200 may be cultured in the structured surface wells 2115 formed by the substrate 2110 in the first chamber 2152, and the second population 2202 of cells is the second. It can be cultured on the porous membrane 2500 in the chamber 2154 of 2.

The devices shown in FIGS. 18-20 include a first port 2162 communicating with the first chamber 2152 and a second port 2164 communicating with the second chamber 2154. In additional embodiments, the first chamber 2152 or the second chamber 2154 has additional ports (exit ports, not shown) as needed to allow liquid to flow through the chambers. Can be done. Ports 2162, 2164 can be, for example, ports similar to the ports illustrated and discussed with respect to FIGS. 21-23 below (eg, port 2160). Ports 2162, 2164 can be on the same side of the device 2100, on both sides, as shown, or provide different access to the chamber or the flow through the chambers 2152, 2154. To do so, it may be oriented in any other suitable manner.

In FIG. 18, the device is shown to be operated with no upper space (filled with cell culture medium to the limit). In FIGS. 19-20, the device is shown to be operated with an upper space (not filled with culture medium to the limit). Due to the porosity of the support 2500, the chamber 2152 remains filled with the culture medium, while the chamber 2154 can be operated with or without an upper space. Due to the gas permeable nature of well 2115, the height of the medium above cells 2200 may not be a significant concern, for example, as mentioned above. However, if the enclosure is otherwise not gas permeable, it may be desirable to limit the height of the medium above the cells cultured on the porous membrane support 2500. As shown in FIG. 20, the porous membrane support 2500 can form a substrate having an array of microwells, eg, as described above.

According to the embodiments shown in FIGS. 18-20, co-culture of two or more populations of cells is planned. For example, a first population of cells can reside in the first chamber 2152, while a second population of cells can reside in the second chamber 2154. These cell populations can be separated by a permeable membrane 2500. This membrane allows chemical communication between a first population of cells and a second population of cells. In embodiments, one or both of these cell populations can be spheroids. For example, as shown in FIG. 20, a first population of spheroid cells 2200 can grow in a first chamber 2152 that forms spheroids due to the spheroid-inducing geometry of the cell culture substrate. Along with this, a second population of spheroid cells 2202 that form spheroids due to the spheroid-inducing geometry of the cell culture substrate present in the second cell culture chamber is also in the second chamber 2154. Can multiply. Through the porous membrane 2500, the second population of spheroid cells 2202 is chemically communicated with the first population of spheroid cells 2200. In this way, it is possible to co-culture two separate populations of spheroid cells while allowing two separate populations of spheroid cells to be chemically communicated with each other. Alternatively, in embodiments, as shown in FIG. 18, the first population of cells may be spheroid cells proliferating within the first cell culture chamber 2152, a second population of cells that are not spheroid cells. (Because the second cell culture chamber does not have a spheroid-inducing geometry) can grow in the second cell culture chamber 2154, while the presence of the porous membrane 2500 is the first and second of the cells. Make groups of cells chemically communicated with each other. As shown in FIG. 19, a second population of cells may be proliferated in the presence of upper space, or as shown in FIG. 18, a second population of cells is absent of upper space. It may be propagated below. Similarly, the upper space may or may not be present in the first cell culture chamber. Alternatively, the spheroid-inducing geometry may or may not be present in the first cell culture chamber. Those skilled in the art will recognize that many combinations of these features may be desirable, depending on the cell culture requirements of the user.

In some embodiments, the housing of the device is gas permeable. As an example, a gas permeable film or bag can form at least a portion of the housing.

In some embodiments, the one or more wells are configured to propagate a single spheroid of defined dimensions, at least in part, based on their defined dimensions and shape. Spheroids can be extended to the dimensional tolerances imposed by the geometry of the wells in which they are cultured. For example, each well may include a microwell or cell culture volume that allows the spheroid to grow to a certain diameter. In other words, the geometric dimensions of the microwell or cell culture volume can force the growth of spheroids so that the diameter of the spheroids reaches and remains at its maximum. Production of spheroids of consistent size can result in tissue-like, non-expandable spheroids, which can be ideal for improving the reproducibility of assay results. Consistent spheroid production can occur as a result of various shaped and sized volumes (eg, microwells or cell culture volumes) defined by the interior of one or more wells. For example, the microwell or cell culture volume is about 200 micrometers to 500 micrometers or any of the above values (eg, 100 micrometers to 200 micrometers, 100 micrometers to 500 micrometers, or 200 micrometers). It can have diameter dimensions ranging from about 100 micrometers to about 700 micrometers), such as (meters to 700 micrometers). Microwells or cell culture volumes can have depths in the range of about 50 micrometers to about 700 micrometers, such as from about 100 micrometers to 500 micrometers, or any range within the values described above.

FIG. 19 is a side view showing an additional embodiment of a cell culture apparatus including a porous membrane support. FIG. 20 is a side view showing an additional embodiment of a cell culture apparatus comprising a porous membrane support showing co-culture of cells.

With respect to FIG. 21, an embodiment of a cell culture apparatus 1400 having a plurality of stacked cell culture compartments 1410A, 1410B, 1410C is shown. Each cell culture compartment may comprise a substrate having an array of microwells as described herein. The device 1400 comprises a packed manifold 1430 having an opening 1435 through which the cell culture medium can be introduced and removed. The filled manifold 1430 has a plurality of openings (not shown). Each cell culture compartment 1410A, 1410B, 1410C has at least one fluid communication with the opening of the manifold 1430 so that the cell culture medium introduced through the opening 1435 can flow into the cell culture compartments 1410A, 1410B, 1410C. It has two openings (not shown). If the device 1400 is oriented to the cell culture position, the opening 1435 may be covered with a cap (not shown) or the like. The device 1400 also comprises an aerated manifold 1420 defining an opening 1425 through which air, metabolic gas, etc. can flow, if necessary. The vented manifold 1420 comprises a plurality of openings (not shown). Each cell culture compartment 1410A, 1410B, 1410C communicated with the opening of the manifold 1420 so that the cell culture metabolic gas could be exchanged between the inside of the cell culture chamber and the outside 1400 of the device through the opening 1425. It has at least one opening (not shown). If the device 1400 is oriented to the cell culture position, the opening 1425 may be covered with a ventilation cap (not shown), a filter (not shown), or the like.

Next, with reference to FIG. 22, a cross-sectional view of the cell culture apparatus 1400, which may be the type of apparatus shown in FIG. 21, is shown. The apparatus 1400 has a plurality of stacked cell culture compartments 1410A, 1410B, 1410C, each having a substrate 1110 defining a structured surface with an array of gas permeable wells described above. In the embodiment shown in FIG. 22, with the exception of the top compartment 1410A in the stack, each compartment (eg, 1410B, 1410C) is defined by a second main surface of a structured substrate 1110 in an adjacent compartment. Has an upper surface of 1450. For example, the second main surface of the substrate 1110 of the compartment 1410B serves as the upper inner surface of the compartment 1410C. Thus, the interiors of adjacent compartments communicate with each other through a common substrate 1110 that forms a structured surface / top surface at the bottom. The top compartment 1410 has an top inner surface 1450 formed by the top, which can be a plate.

The interior of a compartment (eg, compartments 1410A, 1410B, 1410C) is imaged by a substrate having an array of microwells (eg array 100 shown in FIG. 9), a second main surface outside the substrate of the compartment. It is defined by an upper surface formed and one or more side walls 1440. The one or more side walls 1440 communicate with the outside of the device through one or more openings 1425, 1426 defined by the manifold 1420, and the ventilation openings 1442 communicating with the ventilation column 1429 defined by the manifold 1420. Has. The aeration opening 1442 defines the maximum volume of cell culture medium 1300 that may be present inside the cell culture chamber when the device is oriented towards cell culture (eg, as shown in FIG. 22). The volume of cell culture medium in the compartment can be less than maximum. The vent opening 1442 also defines the minimum upper space volume inside the cell culture compartment. The volume of the upper space inside the compartment can be greater than the minimum upper space volume (if the cell culture medium volume is less than the maximum medium volume).

Thus, cells cultured in structured surface wells defined by a substrate 1110 in a chamber (eg, chamber 1410B) above an adjacent chamber (eg, chamber 1410C) are gas permeable wells. Gas communicates with the upper space 1441 of an adjacent chamber (eg, chamber 1410C) via. The upper space 1441 communicates with a column 1429 defined by a manifold, which communicates with the outside of the device through one or more openings 1425, 1426.

A voluntary filter 1427 can be introduced into the opening 1425 or cap 1422 to vent the metabolic gas. Having a vent at the bottom of the device can be advantageous in some embodiments. For example, carbon dioxide, a metabolic waste gas, is denser than the atmosphere and tends to form the highest concentration gradient at the bottom of a culture device that does not have vents at the bottom. Thus, the presence of vents at the bottom of the device (eg, formed by the vent columns 1429 and openings 1426) can facilitate the transfer of waste carbon dioxide out of the device.

Next, with reference to FIG. 23, a cross-sectional view of the cell culture apparatus 1400, which may be the type of apparatus shown in FIG. 21 and may be part of the apparatus shown in FIG. 22 is shown. Unless each reference number in FIG. 23 is explicitly discussed, refer to the description of similarly numbered components described above with respect to FIG. In an exemplary embodiment, the interior of a compartment (eg, compartments 1410A, 1410B, 1410C) is a structured surface inside the substrate 1110, an upper surface defined by the outer surface of the substrate of the compartment, and 1 It is defined by one or more side walls 1440. One or more side walls 1440 are imaged by a filled manifold 1430, which communicates with the outside of the device through an opening 1435 defined by a manifold 1430, which can be covered by a cap 1432 when the device is oriented to cell culture. It has an opening 1443 that communicates with the column 1439 formed. The cell culture medium 1300 can be introduced into or removed from the cell culture compartment (eg, compartments 1410A, 1410B, 1410C) via the column 1439 and opening 1435 by operating the device.

Next, referring to FIGS. 24A and 24B, the role of forming cell culture compartments (eg, compartments 1410A, 1410B, 1410C shown in FIGS. 21-23) when multiple trays are stacked on top of each other. A schematic bottom view (24A) and a schematic perspective view (24B) of the tray 1415 are shown. Tray 1415 comprises side walls 1440A, 1440B, 1440C, 1440D extending from substrate 1110 defining a substrate with an array of microwells. In some embodiments, the tray comprises a single surrounding sidewall (not shown). A partially heighted wall 1472 connects to side walls 1440A and 1440D. Partial height walls 1472 and side walls 1440A, 1440D surround and define the ventilation opening 1442. When the cell culture device is assembled from stacked trays, the metabolic gas inside the chamber formed by the tray 1415 can flow through the opening 1442 over the partial wall 1472. The vent openings 1442, partial walls 1472 and connecting sidewalls of the stacked trays 1415 may form at least a portion of the vent column (eg, vent column 1429 shown in FIG. 22).

Tray 1415 also includes a partially heighted wall 1473 connected to sidewalls 1440A and 1440B. Partial height walls 1473 and side walls 1440A, 1440B surround and define the filling opening 1443. When the cell culture device is assembled from the stacked trays, the culture medium is operated by the assembled device, over the partial wall 1473, through the filling opening 1443, and inside the chamber formed by the tray 1415. Can be introduced into or removed from it. The filling opening 1443, the partial wall 1473 and the connecting side walls of the stacked tray 1415 may form at least a portion of the filling column (eg, the filling column 1439 shown in FIG. 23).

The height of the partial wall 1473 defines the maximum height and volume of the cell culture medium that can be contained within the cell culture compartment formed by the tray 1415. The top of the partial wall 1473 can be at any suitable distance from the substrate 1110 forming the structured surface. In some embodiments, the distance is 5 mm or more, such as 6, 7, 8, 9, 10, 12, 15, or 20 mm, including a range between any two of the following: The height of the cell culture medium above the cells is gas exchange, as the gas permeable wells on the structured surface of the cell culture device assembled from such trays communicate with the outside of the device. Can be higher than the height available with conventional cell culture equipment, which occurs primarily through the cell culture medium.

The distance from the top of the partial wall 1472 to the substrate 1110 can be equal to or greater than the distance from the top of the partial wall 1473 to the substrate 1110. Therefore, if the compartment is overfilled with culture medium, the excess medium is drained through the filling opening 1443 rather than through the aeration opening 1442. In embodiments that employ a bottom filter (such as filter 1427 in FIG. 22) for the aeration column, the bottom filter will not be contaminated with medium. Of course, proper operation of the assembled device will also prevent media contamination of the bottom filter.

Next, with reference to FIG. 25, a schematic side view of an embodiment of the cell culture apparatus 1400 having a plurality of stacked cell culture compartments 1410A, 1410B, 1410C is shown. Each compartment may include a substrate 1110 that defines the structured surface described above. The apparatus 1400 comprises a spacer 1500, which is positioned adjacent to a substrate 1110 forming a structured surface and provides a passage for airflow to the outside of the chamber, the passage being ". It is referred to as a "conduit space" (eg, conduit spaces 1460A, 1460B, 1460C). Since the structured surface defines gas permeable wells in which cells can be cultured, metabolic gas passes through the wells and is defined by spacers 1500 to 1400 outside the device, conduit spaces 1460A, 1460B. , Can be exchanged for 1460C. The apparatus 1400 also comprises a manifold 1430 that defines an opening through which the cell culture medium can be introduced or removed. The manifold 1430 has a plurality of openings (not shown). Each cell culture compartment 1410A, 1410B, 1410C has at least one opening in fluid communication with the opening of the manifold 1430 so that the cell culture medium introduced through the manifold 1430 can flow into the cell culture compartments 1410A, 1410B, 1410C. (Not shown).

The bottom of the cell culture apparatus 1400 in FIG. 25 comprises a plate 1510 on which the spacer 1500 is placed. In embodiments, the plate 1510 and spacer 1500 are unified parts, such as molded parts. Such plates can form the top surface of other cell culture compartments (eg, compartments 1410A, 1410B, 1410C). For each compartment, one or more side walls 1440 extend from the substrate 1100, which defines the structured surface, to the top surface, which top surface can be formed from a plate with spacers. An opening (not shown) that communicates with the opening (not shown) of port 1430 can be defined by a side wall.

The stacked cell culture trays or chambers can be assembled in any suitable manner. For example, such components can be bonded using welding techniques (eg, heat, laser, long wave IR or ultrasonic welding, etc.), bonding, solvent bonding and the like.

In some embodiments, the structured surface is attached to the bag. Bags suitable for cell culture can be formed from film by heat sealing, laser welding, adhesive application, or any other method known in the field of inflatable bag manufacturing. The walls of the bag or parts thereof may have a thickness that allows efficient movement of gas across the walls. It should be understood that the desired thickness can vary depending on the material forming the wall. As an example, the wall or the film forming the wall can be about 0.02 mm to 0.8 mm thick. The bag can be made from any material suitable for cell culture. In various embodiments, the bag is optionally formed from a transparent material to allow visual inspection of the cells cultured in the bag. Examples of optionally transparent, gas permeable materials that can be used to form bags include polystyrene, polycarbonate, poly (ethylene-vinyl acetate), polysulfone, polymethylpentene, polytetrafluoroethylene (PTFE) or Compatible fluoropolymers, silicone rubbers or copolymers, poly (styrene-butadiene-styrene); or polyolefins such as polyethylene or polypropylene; or combinations of these materials.

The cell culture apparatus described herein can be used to culture cells in structured surface wells. As mentioned above, the cell culture medium in the device can be present at any suitable height above the cells. In some embodiments, the height of the cell culture medium in the device above the cells (eg, above the top or bottom of the well) is about 5 mm or greater. In some embodiments, the height of the cell culture medium in the device above the cells (eg, above or below the well) is about 6 mm or more, about 7 mm or more, about 8 mm or more, about 9 mm or more, or about 10 mm. That is all.

Due to the gas permeability of the wells, such a height of the cell culture medium can be used to keep the cells healthy. Therefore, cells cultured in the apparatus described herein can be cultured for long periods of time with cell culture medium of such height. For example, cells can be continuously cultured with medium of such height for 24 hours or longer, 48 hours or longer, 72 hours or longer, 96 hours or longer, or until the medium is replaced.

In embodiments where the wells are non-adhesive to the cells, the cells can be harvested by flipping the device to allow the cells to move out of the wells by gravity.

In some embodiments, the porous membrane is in the same device, but separate from the first cell type cultured on a structured surface that is gas-communicating with the outside of the device, the second cell. To support the growth of the type (or to support the growth of additional cells of the same type), they are placed in the cell culture apparatus described herein. In some embodiments, stem cells are cultured on a structural surface with gas permeable wells communicating with the outside of the device, and supporting cells are cultured on a porous membrane. Of course, using such a device, any other desired combination of cells and compartments can also be employed.

The porous membrane can be placed within the housing of the cell culture apparatus to compartmentalize the housing into two growth chambers. Preferably, the permeable membrane limits cell migration through the membrane but allows the passage of biomolecules. Examples of materials that can be used to form porous membranes include track-etched membranes or woven or non-woven porous materials. The material of the porous membrane can be treated or coated to make it more adhesive or non-adhesive to the cells. The treatment can be accomplished by any method known in the art, including plasma discharge, corona discharge, gas plasma discharge, ion shock, ionized radiation, and high intensity UV light. The coating can be introduced by any suitable method known in the art, including printing, spraying, condensation, radiant energy, ionization or immersion. The coating may then provide either a shared or non-shared binding site. Such sites can be used to bind moieties such as cell culture components (eg, proteins that promote proliferation or adhesion). In addition, coatings can also be used to enhance the binding of cells (eg, polylysine). Alternatively, the cell non-adhesive coating described above can be used to prevent or suppress cell binding. In some embodiments, the porous membrane has a structured surface with a plurality of wells as described above with respect to a substrate forming a structured surface defining a plurality of gas permeable wells. Can be manufactured as However, in this case, the porous membrane material is formed to have a structured surface.

Gas permeable wells on the structured surface (eg, those mentioned above) allow control of oxygen tension by adjusting the gas concentration in the incubator where the device is placed to allow cell proliferation. .. Permeable membrane supports provide a way to physically separate different cell populations while allowing the transfer of biologically active components.

The porous membrane can be attached to the housing of the device (eg, side walls, etc.) in any suitable manner. For example, the porous membrane can be introduced into the apparatus in a manner similar to the introduction of the substrate forming the substrate defining the structured surface having the plurality of gas permeable wells described above.

In the embodiment shown in FIG. 26, the cell culture apparatus 3100 is a 96-well multi-well plate having wells 3115 within a plate 3111 surrounded by a frame 3113. However, as mentioned above, the cell culture apparatus may have an appropriate number of wells (eg, 3, 6, 12, 96, or any other number of wells may be provided). In the illustrated embodiment, at least a portion of each of the plurality of wells 3115 comprises a substrate having an array of microwells and provides a position for forming the spheroids described above. Almost any type of cell culture device having wells used for culturing cells can form the full volume of the wells in some embodiments, or in some embodiments the cells of the wells. It can be designed by adopting a substrate with an array of microwells capable of forming a sub-volume of culture. In some embodiments, the cell culture apparatus in which the microwell design can be performed using it is a multiwell such as a 96-well multiwell plate, a 384-well multiwell plate, a 1536-well multiwell plate. It can be a plate. In some embodiments, at least the surface of the multiwell plate is gas permeable.

The ability of multiple wells to allow cells to aggregate to form spheroids, as well as the ability to form spheroids within each of multiple wells to maintain consistent dimensions across multiple wells. , Can be achieved in any suitable manner. For example, the plurality of wells 3115 may comprise an array of microwells structured and arranged such as the microwells shown in FIGS. 1-5, or 16 in which cells proliferate as spheroids 500.

In some embodiments, the one or more wells are configured to propagate a single spheroid of defined dimensions, at least in part, based on their defined dimensions and shape. Spheroids can be extended to the dimensional tolerances imposed by the geometry of the wells in which they are cultured. For example, each well may have a microwell or cell culture volume that allows the spheroid to grow to a certain diameter. In other words, the geometric dimensions of the microwells or cell culture volumes can force the growth of spheroids so that the diameter of the spheroids reaches and remains at its maximum. The production of consistently sized spheroids can result in tissue-like non-expansive spheroids, which can be ideal for improving the reproducibility of assay results. Consistent spheroid production can be the result of various shaped and sized volumes (eg, microwells or cell culture volumes) defined by the interior of one or more wells. For example, the microwell or cell culture volume is about 200 micrometers to 500 micrometers or any of the above values (eg, 100 micrometers to 200 micrometers, 100 micrometers to 500 micrometers, or 200 micrometers). It can have diameter dimensions ranging from about 100 micrometers to about 700 micrometers), such as (meters to 700 micrometers). Microwells or cell culture volumes can have depths in the range of about 50 micrometers to about 700 micrometers, such as from about 100 micrometers to 500 micrometers, or any range within the values described above.

Each of the plurality of wells described herein can assist cells deposited therein to form spheroids. Each of the plurality of wells also has a diameter of each spheroid about, eg, 500 micrometers or less, 400 micrometers or less, 300 micrometers or less, 250 micrometers or less, 150 micrometers or less, etc., or any of the above values. Values in this range (eg, 150-250, 150-300, 150-400, 150-500, 250-300, 250-400, etc.) can be limited or enforced. In some embodiments, each of the plurality of wells has an average diameter of all spheroids grown in the plurality of wells, such as about 20% or less, 15% or less, 10% or less, 5% or less. It forms spheroids defined by diameters that differ by no more than 2, 2%, etc., or by any range within the above values.

In the embodiments shown in FIGS. 27-29, an array of microwells 3115 is formed within the substrate 3110. Each of the plurality of wells 3115 may have an upper opening 3118, a bottom surface 3112, and a side wall surface 3120 extending from the top opening 3118 to the bottom surface 3112. In addition, the side wall surface 3120 is so called between the top opening 3118 and the bottom surface 3112 because the reduced area at the bottom of the well looks like the tip of a pen, the nib area 3116 (see FIG. 29). Can be defined. The nib area has a geometric shape that induces spheroids.

Each upper opening 3118 of the plurality of wells 3115 can be utilized as an opening through which cells are seeded into each of the plurality of wells 3115. The top opening 3118 can have a variety of different shapes and dimensions. For example, the upper opening 3118 can be defined by a shape, such as a circle, an ellipse, a square, a rectangle, a hexagon, a quadrilateral, and the like. The top opening 3118 can also be defined by diameter dimensions (depending on shape, eg diameter, width, etc.). The diametrical dimension of the top opening 3118 can be defined as the distance across the top opening at the widest point. The diameter dimension of the upper opening 3118 is about, for example, 300 micrometers or more, 500 micrometers or more, 800 micrometers or more, 1000 micrometers or more, 1500 micrometers or more, 2000 micrometers or more, or 7000 micrometers or less. It can be 6000 micrometers or less, 4000 micrometers or less, 2500 micrometers or less, 1700 micrometers or less, 1200 micrometers or less, etc., or any of the above values.

The bottom surface 3112 of each of the plurality of wells 3115 may prompt to allow cell culture above or above them. The bottom surface 3112 can have a variety of different shapes and dimensions. For example, the bottom surface 3112 can be circular, hemispherical, flat, conical, or the like. As a further example, the bottom surface 3112 can also be defined by shapes such as circles, ellipses, squares, rectangles, hexagons, quadrilaterals and the like. As shown in FIGS. 27-29, the bottom surface 3112 is flat and defined by diameter dimensions. The diameter dimension of the bottom surface is about, for example, 0 micrometer or more, 50 micrometers or more, 75 micrometers or more, 100 micrometers or more, 200 micrometers or more, 275 micrometers or more, or 700 micrometers or less, 500 micrometers or more. It can be meters or less, 400 micrometers or less, 300 micrometers or less, 250 micrometers or less, 150 micrometers or less, or any of the above values. For a bottom surface 3112 having a round bottom or similar surface (eg, hemispherical, conical, etc.) with the bottom positioned at the lowest point, the diameter dimension of the bottom surface 3112 is considered to be zero. In some embodiments, the diameter dimension of the top opening 3118 is greater than the diameter dimension of the bottom surface 3112. In another embodiment, the diameter dimension of the top opening 3118 is equal to the diameter dimension of the bottom surface 3112.

In some embodiments, the bottom surface 3112 of each of the plurality of wells 3115 can be uniformly constructed with a substrate having a microarray of wells (see also FIG. 29). In other embodiments, the bottom surface 3112 can be made from a material different from the material used to form the substrate 3110. The various methods of producing multiple wells are further described below. The bottom surface 3112 or side wall surface 3120 may be gas permeable to aid the supply of oxygen to cells or spheroids 3130 cultured in wells 3115. In some embodiments, the substrate 3150 defining the bottom surface 3112 can be a gas permeable substrate. In some embodiments, the substrate 3150 may include a gas permeable film. The gas permeability of the bottom surface 3112 to the outside depends in part on the material of the bottom surface 3112 and the thickness of the bottom surface 3112. For example, the gas permeability of a well is incorporated herein by reference in its entirety, unless inconsistent with the present disclosure, filed October 29, 2014, "GAS PERMEABLE CULTURE FLASK". ) ”, Which can be described in US Provisional Patent Application No. 62/072,088.

The nib region 3116 (see FIG. 29) can be defined by the side wall surface 3120 between the top opening 3118 and the bottom surface 3112. The location of the nib region 3116 may be defined by other components of the well. For example, the nib region 3116 can be defined by a diameter dimension of 3144 that crosses the side wall surface 3120. The diameter dimension 3144 of the nib region 3116 can be defined as the distance across the side wall surface 3120 in the nib region 3116. The pen tip area 3116 is about, for example, 50 micrometers or more, 100 micrometers or more, 200 micrometers or more, 300 micrometers or more, 400 micrometers or more, 550 micrometers or more, or 800 micrometers or less, 700 micrometers. It can be defined by a diameter dimension of 3144, such as meters or less, 600 micrometers or less, 500 micrometers or less, 450 micrometers or less, 350 micrometers or less, or any of the above values. The pen tip area 3116 is also about, for example, 50 micrometers or more, 100 micrometers or more, 150 micrometers or more, 250 micrometers or more, 350 micrometers or more, 450 micrometers or more, or 800 micrometers or less, 700. It can also be defined by a height of 3142 from the bottom surface 3112, such as micrometer or less, 600 micrometers or less, 500 micrometers or less, 400 micrometers or less, 300 micrometers or less, or any of the above values. .. The height 3142 can be measured from the lowest point of the bottom surface 3112. In such an embodiment, the total volume of wells 3115 is the cell culture volume 3140.

The diameter dimension of the top opening 3118 can be greater than or equal to the diameter dimension 3144 of the pen tip region 3116. The diameter dimension 3144 of the pen tip region 3116 can be greater than or equal to the diameter dimension of the bottom surface 3112. It can also be stated that the diameter dimension of the bottom surface 3112 is less than or equal to the diameter dimension of the pen tip region 3116, or the diameter dimension 3144 of the pen tip region 3116 can be less than or equal to the diameter dimension of the upper opening 3118. In some embodiments, the top opening 3118 can be the nib region 3116.

The side wall surface 3120 of each of the plurality of wells extends from the top opening 3118 to the bottom surface 3112. The side wall surface 3120 may include an upper side wall surface 3124 and a lower side wall surface 3122. The upper side wall surface 3124 may be defined between the upper opening 3118 and the nib region 3116. The lower side wall surface 3122 may be defined between the pen tip region 3116 and the bottom surface 3112. In some embodiments, each side wall surface 3120 of the plurality of wells 3115 may define a cell non-adhesive surface. The non-adhesive surface promotes the growth of cells to spheroids 3130 in the previously mentioned cell culture volume 3140. The cell non-adhesive upper sidewall surface 3124 can facilitate the sedimentation of seeded cells within the cell culture volume 3140. Regardless of whether the upper side wall surface 3124 is cell non-adhesive, the upper side wall surface 3124, in some embodiments, causes the cells seeded in the wells to settle into the nib region 3116. It can be configured to form a cell culture volume of 3140 as a result of gravity.

In some embodiments, the upper side wall surface 3124 and the lower side wall surface 3122 can be defined by shapes such as paraboloids, cones, steps, various angles, curves and the like. The upper and lower side wall surfaces 3124 and 3122 may have the same or different shapes. In some embodiments, the side wall surface 3120 may have an inflection point 3121 at the location where the upper and lower side wall surfaces 3124 and 3122 meet (eg, shown in FIG. 29). In other embodiments, the side wall surface 3120 may have an inflection point 3121 where the surfaces of the upper and lower side walls 3124 and 3122 meet.

In some embodiments, a portion of the side wall 3120 adjacent to the bottom surface 112 may be perpendicular to the bottom surface 3112 or at an angle to the bottom surface 3112. A portion of the side wall 3120 adjacent to the bottom surface 3112 may be described as a lower side wall surface 3122. The angle at which a part of the side wall 3120 intersects the bottom surface 3112 is, for example, 90 degrees or more, 92 degrees or more, 95 degrees or more, 100 degrees or more, or 110 degrees or less, 105 degrees or less, 102 with respect to the bottom surface 3112. It can be defined in the range of degrees or less, 97 degrees or less, or any of the above values. In some embodiments, the diametrical dimension across the side wall surface 3120 can be described as increasing from the bottom surface 3112 towards the top opening 3118. The geometry of the sidewalls can be such that the cells are sufficiently submerged within each of the plurality of wells 3115.

For a bottom surface 3112 that does not have a flat surface, the angle of the side wall 3120 is considered to be relative to the tangential plane of the bottom surface of the bottom surface 3112. In other embodiments, the virtual plane can also be defined as coplanar with the top opening 3118, regardless of whether the virtual plane is tangential to the bottom.

A partial combination of the bottom surface 3112, the nib region 3116 and the side wall surface 3120 can define a cell culture volume 3140. A portion of the side wall surface 3120 that defines the cell culture volume 3140 can also be described as the lower side wall surface 3122. Cells are not limited to being cultured only within a cell culture volume of 3140. However, cells deposited within each of the plurality of wells 3115 can aggregate within the cell culture volume 3140 to form and proliferate the spheroid 3130. Also, the dimensions of the spheroid 3130 may be the result of the shape and dimensions of the cell culture volume 3140. For example, each cell culture volume 3140 of the plurality of wells 3115 has a spheroid 3130 of about, for example, 500 micrometers or less, 400 micrometers or less, 300 micrometers or less, 250 micrometers or less, 150 micrometers or less, or the like or described above. It is configured to grow to a diameter in any range within the value of. In some embodiments, the cell culture volume 3140 of each of the plurality of wells 3115 is approximately, for example, 20% or less, 15% or less, with respect to the average diameter of all spheroids 3130 grown in the plurality of wells 3115. It forms spheroids 3130, which are defined by diameters that differ by 10% or less, 5% or less, 2% or less, etc., or by any range within the above values.

A partial combination of the top opening 3118, the nib area 3116 and the side wall surface 3120 defines a second volume 3145. A portion of the side wall surface 3120 that defines the second volume 3145 can also be described as the upper side wall surface 3124. The second volume 3145 may be larger than the cell culture volume 3140. For example, the second volume 3145 is about 100% or more, 200% or more, 500% or more, 1,000% or more, 10,000% or more, 100,000% or more, 200,000% of the cell culture volume 3140. It can be the above, or any of the above values. As an example, a 96-well plate is defined by a cell culture volume defined by a volume of 0.1 microliter and a volume of 200 microliters, resulting in 2,000 times larger than the cell culture volume. It can have a second volume that can produce a volume.

One embodiment of the cell culture apparatus 3100 is shown in FIG. As shown in FIG. 27, the side wall surface 3120 of each of the plurality of wells 3115 gradually narrows from the top opening 3118 to the bottom surface 3112. In particular, the side wall surface 3120 is approximately perpendicular to the top opening 3118, but from the top opening 3118 at a slight angle that reduces the diameter dimension across the side wall surface 3120 as the side wall surface 3120 extends toward the bottom surface 3112. It is extending. At a point 3123 along the side wall surface 3120 between the top opening 3118 and the nib area 3116, the angle of the side wall surface 3120 further increases the diameter dimension across the side wall surface 3120 as the side wall surface 3120 extends towards the bottom surface 3112. Change to lower. In the nib region 3116, the angle of the side wall surface 3120 also changes and extends towards the bottom surface 3112. This last portion of the side wall surface 3120 is sometimes referred to as the lower side wall surface 3122. As shown in FIG. 27, in the embodiment, the lower side wall surface 3122 may be slightly angled from the vertical with respect to the bottom surface 3112, and the diameter dimension across the side wall surface 3120 is such that the side wall surface 3120 is the bottom surface 3112. It decreases as it extends to. The spheroid 3130 is shown to be positioned within the lower sidewall surface 3122 and with respect to the bottom surface 3112 (ie, within the nib region 3116). The lower sidewall surface 3122 may limit or limit the dimensions in which the spheroid 3130 can grow.

One embodiment of the cell culture apparatus 3100 is shown in FIG. As shown in FIG. 28, the side wall surface 3120 of each of the plurality of wells 3115 gradually narrows from the top opening 3118 to the bottom surface 3112. Specifically, the side wall surface 3120 first extends from the top opening 3118 at an angle perpendicular to the top opening 3118 and then extends toward the nib region 3116 along a parabolic trajectory. In the nib region 3116, the angle of the side wall surface 3120 changes and extends towards the bottom surface 3112. This last portion of the side wall surface 3120 is sometimes referred to as the lower side wall surface 3122. As shown in FIG. 29, the lower side wall surface 3122 is slightly angled from perpendicular to the bottom surface 3112, and the diametrical dimension across the side wall surface 3120 decreases as the side wall surface 3120 extends toward the bottom surface 3112. The spheroid 3130 is shown to be positioned within the nib region 3116 within the lower sidewall surface 3122 and with respect to the bottom surface 3112. The lower sidewall surface 3122 may limit or limit the dimensions in which the spheroid 3130 can grow. The nib area has a geometric shape that induces spheroids.

Next, focusing on FIG. 30, in some embodiments, the cell culture apparatus 3650 may include a bottom plate 3610 and one or more side walls 3620, as shown in FIG. The bottom plate 3610 can define the main surface 3611 and one or more side walls 3620 can extend from the bottom plate 3610. The bottom plate 3610 can be formed entirely or partially from a substrate having an array of microwells 3615. FIG. 30 shows that the bottom plate can have one of a plurality of arrays of microwells 3615. That is, each of the regions identified as an array of microwells 3615 shown in FIG. 30 may contain a much smaller array of microwells. In an embodiment, the cell culture device 3650 may also include a plurality of wells 3615 formed on the main surface 3611 of the bottom plate 3610. Each well of the plurality of arrays of microwells 3615 can define the microwells or cell culture volumes described above that promote or induce the growth of spheroids. The main surface 3611 of the bottom plate 3610 and one or more side walls 3620 define the volume of the reservoir. The reservoir plates described herein allow the addition of culture medium, the excess of which is typically used to fill the individual shallow wells of the microwell plate and cells in different wells that communicate with the fluid. Allows culture.

In some embodiments, one or more side walls 3620 can be extended further from the bottom plate 3610 (eg, side wall height) than some current commercially available cell culture devices, and the reservoir is medium. Allows the retention of more medium than the normal volume of. Larger capacity conditions for the reservoir may allow excess culture medium to be added to the reservoir so that the spheroids do not have to rely solely on the amount of medium in each individual well. In other words, spheroids may not need to be supplied with cell culture medium as often as the spheroids grow in standard microplate wells. As shown in FIG. 30, since the cell culture medium in the reservoir communicates with all wells in the reservoir, nutrients and biotransformers can be exchanged throughout the cell culture medium.

In some embodiments, the cell culture assembly 3600 may comprise a cell culture apparatus 3650 and a fluid permeable mesh 3670. The fluid permeable mesh 3670 can be placed on top of the well 3615 after the cells have been seeded into the wells. Cell culture medium that communicates well among multiple wells 3615 can be sequestered and replaced during a manual batch feeding process without disturbing the cells in the wells. Since the cells can be non-adhesive to the surface of the wells in some embodiments, it can be difficult to replace the cell culture medium without disturbing or losing the spheroids. However, by using the mesh 3670 described above, such difficulties can be alleviated. For example, the combination of a cell culture device and a fluid permeable mesh is incorporated herein by reference in its entirety, as long as it is not inconsistent with the present disclosure, according to the invention of the "RESERVOIR SPHEROID PLATE". It may be as described in US Provisional Patent Application No. 62 / 072,103, filed October 29, 2014 by name.

It will be appreciated that the wells 3615 of the cell culture apparatus 3650 described herein can be of any size, shape or configuration. In some embodiments, the wells are formed from a hexagonal close-packed well structure, as shown in FIG. Reservoir plates with a structured surface with tightly packed small volume wells, as small volume wells will require frequent replacement of cell culture medium without additional reservoir volume. The device can be particularly advantageous.

The reservoir plates described herein allow, for example, the addition of culture medium, the excess of which is typically used to fill the individual shallow wells of the microwell plate and within different wells. Allows cultured cells to communicate with fluid.

As shown in FIG. 31, the cell culture apparatus 4100 may include a bottom plate 4110 and one or more side walls 4120. The bottom plate may define the main surface and one or more side walls 4120 may extend from the bottom plate. The combination of the bottom plate and one or more side walls can define a reservoir. The cell culture apparatus may also include, in whole or in part, a substrate having an array of microwells 4115 formed on the main surface of the bottom plate. Each well of the plurality of wells in the microwell array can define a top opening and a bottom. The top opening may be coplanar with the main surface and the bottom may be positioned below the main surface, i.e. the bottom is oriented opposite to the direction in which one or more side walls extend from the bottom plate Can be done. The top plate (not shown) can be placed on the desired reservoir while incubating the cells.

In some embodiments, the one or more sidewalls can extend farther from the bottom plate than typical, thus allowing the reservoir to retain a larger volume than the normal volume of the medium. Larger capacity conditions for the reservoir may allow excess culture medium to be added to the reservoir so that the spheroids may not have to rely solely on the amount of medium in each individual well. In other words, spheroids may eliminate the need to supply cell culture medium as often as the spheroids grow in standard microplate wells. As shown in FIG. 31, since the cell culture medium in the reservoir communicates with all wells in the reservoir, nutrients and biotransformers can be exchanged throughout the cell culture medium.

In some embodiments, the cell culture assembly 4200 is described herein. The assembly may include a device 4100 (eg, a device as shown and described with respect to FIG. 31) and a fluid permeable mesh 4570. The fluid permeable mesh 4570 can be placed on top of the well 4115 after the cells have been seeded into the wells. Commonly communicated cell culture media can be sequestered and replaced during a manual batch feeding process without disturbing the cells in the wells.

In some embodiments (eg, shown in FIG. 32), the frame 4560 can be connected to the mesh 4570 as shown. The frame 4560 may be configured to keep the mesh 4570 in place on the first well 4515 of the wells. In some embodiments, the mesh 4570 may be configured to be placed on the upper edge of the side wall 4120 of the device 4100. The frame 4560 can engage one or more side walls 4120 via a tight fit, snap fit, or any other suitable mechanism for holding the mesh onto the main surface of the plate 4110. In some embodiments, the user can manually hold the frame 4560 in place so that the mesh is held on the main surface of the plate 4110 on the well 4115.

The fluid permeable mesh 4570 can be formed from any suitable material. In some embodiments, the fluid permeable mesh defines the pores. The pores can be of any suitable size. In some embodiments, the pores define an average pore size in the range of 10 micrometers to 100 micrometers. In some embodiments, the pores define an average pore size of 40 micrometers or less. Preferably, the pores of the mesh are small enough to prevent spheroids from passing through the mesh.

In some embodiments, the mesh is incorporated herein by reference in its entirety, as long as it is not inconsistent with the present disclosure, eg, US Provisional Patent Application No. 62/072094, assigned to the assignee of the invention. It is described in the book.

In some embodiments, instead of manually replacing the cell culture medium, the reservoir plate described herein is as a perfusion device that allows the cell culture medium to flow over the reservoir on the main surface of the well. Can be manufactured.

For example and with reference to FIG. 33, in the cell culture apparatus 4100 described in FIG. 31 and discussed with respect to FIG. 31, one or more side walls form an inlet 4140 and one or more side walls form an outlet 4145. Can be adapted to do so. The cell culture fluid can be perfused throughout the reservoir from inlet to outlet. The device shape factor shown in FIG. 33 may be an open top shape factor or a closed top shape factor. If the Scherrer equation is an open top, inserts containing the frames and meshes discussed with respect to FIG. 2 would otherwise move cells such as spheroids out of the wells at high perfusion rates. It can be used to hold cells in well 4115.

The cell culture apparatus described herein can be manufactured in any suitable manner. In various embodiments, the method of making a cell culture device comprises molding a polymeric material, or any other suitable material described herein, to form the cell culture device. The polymeric material can define multiple wells of the cell culture apparatus. Each of the plurality of wells can define a top opening, a bottom surface, and a side wall surface extending from the top opening to the bottom surface. The side wall surface can also define the nib area between the top opening and the bottom surface. The polymeric material can be poured into a mold with pins so that the polymeric material formed around the pins has the properties of the multiple wells described herein.

In some embodiments, the polymeric material is coated and molded onto a substrate to form a cell culture device. The substrate defines the bottom surface, and the combination of the polymer material and the substrate defines multiple wells. The polymeric material can be poured into a mold with pins so that the polymeric material formed around the pins has the properties of the multiple wells described herein.

In some embodiments, regardless of how the cell culture apparatus described herein is manufactured, the side wall surface of each of the plurality of wells is non-adhesive to the cells further described herein. Can be coated with the following materials.

In some embodiments, the well comprises a portion of the side wall or various features added to the side wall. Features of such substrates may be directly injection molded, or they may be embossed onto the formed substrate. The characteristic material can be any polymer, polymer blend, copolymer, glass, metal, or any other material described herein or understood in the art.

The devices, wells, sidewalls, well bottoms, and other features described herein are made of any suitable material. Preferably, the material intended for contact with the cell or culture medium is compatible with the cell and medium. Typically, the cell culture components are formed from a polymeric material. Examples of suitable polymer materials are polystyrene, polymethyl methacrylate, polyvinyl chloride, polycarbonate, polysulfone, polystyrene copolymers, fluoropolymers, polyesters, polyamides, polystyrene butadiene copolymers, fully hydride styrene polymers, polycarbonate PDMS. Examples thereof include copolymers and polyolefins such as polyethylene, polypropylene, polymethylpentene, polypropylene copolymers and cyclic olefin copolymers.

In embodiments, the inner surface of the well is non-adhesive to the cells. The wells may be formed from a non-adhesive material or may be coated with a non-adhesive material to form a non-adhesive surface. Examples of non-adhesive 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 mixtures thereof. In some embodiments, for example, a combination of two or more of non-adhesive wells, well geometry, and / or gravity causes cells cultured in the wells to self-assemble into spheroids. Is triggered. Some spheroids maintain differentiated cell function with a greater in vivo-like response compared to cells proliferated in a monolayer. In embodiments where the wells are non-adhesive to the cells, the cells can be harvested by turning the device upside down so that the cells can move out of the wells by gravity.

In some embodiments, surface modification of the material is used to achieve the desired properties. Such modifications include surface chemistry modifications and mechanical properties include biological coatings (eg, Matrigel ™, collagen, laminin, etc.) and synthetic coatings (eg, Synthemax®, silicones). It can be used using (hydrogel, etc.). Other surface modifications to the material (eg, within wells or microstructures) are within the scope of the invention.

Substrates with structured surfaces as described herein can be assembled into cell culture chambers or trays in any suitable manner. For example, the structured surface of a cell culture chamber or tray and one or more other components can be molded as a single part. In some embodiments, the structured surface or portion thereof is coated and molded to form a bottom and one or more components, the structured surface being one or more of cell culture devices. Other components include welding (eg, heat, laser, long wave IR or ultrasonic welding, etc.), bonding, solvent bonding and the like.

In various embodiments, the cell culture system may include two or more components of the cell culture apparatus described above. As an example, the components of the device can be stacked to form a cell culture system. Examples of stacked cell culture systems capable of incorporating the components of the cell culture apparatus described herein are described herein by reference in their entirety, for example, unless they are inconsistent with the present disclosure. (I) US Provisional Patent Application No. 62 / 072,015 filed on October 29, 2014 under the name of the invention "MULTILAYER CULTURE VESSEL"; (ii) "Perfusion bioreactor". US Provisional Patent Application No. 62/072,039, filed October 29, 2014, under the name of the invention "PERFUSION BIOREACTOR PLATFORM".

The cell culture apparatus described herein can be used to incubate the cells in the wells of the apparatus in any suitable manner. For example, a method for culturing cells comprises the step of introducing cells and cell culture medium into one or more of the plurality of wells of the cell culture apparatus described herein. The cell culture medium can be contained only in the cell culture volume or in each of the plurality of wells, including the cell culture volume and the second volume. The method also includes culturing cells in a medium in one or more of a plurality of wells. The step of culturing cells in one or more of the plurality of wells may include the step of forming spheroids in the one or more wells. Spheroids cultured in one or more wells are approximately, for example, 500 micrometers or less, 400 micrometers or less, 300 micrometers or less, 250 micrometers or less, 150 micrometers or less, or any of the above values. It can be defined by the diameter of the range. The diameter of one spheroid is about, for example, 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, etc. or the above-mentioned average diameter of all spheroids grown in a plurality of wells. It can vary in any range within the value.

In some embodiments, the well bottom comprises a concave arched surface or a "cup" geometry, such as a hemisphere, a conical surface with a round bottom, and similar surface shapes, or a combination thereof. .. Wells (eg, microwells) and well bottoms eventually terminate within a spheroid-friendly rounded surface or curved surface, such as a recess, a concave truncated cone undulating surface, or a combination thereof. Can be terminated or bottomed out.

In certain embodiments, the portion of the sidewall and / or the bottom of the well is of varying degrees of opacity / transparency with respect to wavelengths within the visible and / or UV spectrum. For example, an opaque side wall can be combined with a transparent microwell bottom. The transition from opaque to transparent can be gradual or radical.

In some embodiments, the well (eg, a microwell) has a low-adhesive, non-adhesive, or high-adhesive coating on a portion of the well, such as on at least one concave arched surface. Including.

In some embodiments, the device further comprises, for example, a well annex, a well expansion region, or an auxiliary side chamber to receive the tip of a suction pipette. In some embodiments, the well attachment or well extension (eg, side pocket) is, for example, an integral surface adjacent to the well (eg, microwell) and in fluid communication. In some embodiments, the well appendage has a bottom separated from the gas permeable transparent bottom of the well. The well attachment and the second bottom of the chamber well are separated from the transparent bottom of the gas permeable, for example at a higher position or at a relatively higher position. In some embodiments, the second bottom of the well appendage deflects the fluid distributed from the pipette away from the clear bottom to avoid spheroid splitting or disruption.

In certain embodiments, the device is further located inside a portion of the well, inside a portion of the well appendage, or inside both the well and the well appendage, a liner or membrane. It has a porous membrane such as an insert. The porous membrane is located from the first cell material below the bottom of one or both wells near the bottom, to the top of the well, to the top of the well formed by the porous membrane, or to the top of both wells. It can provide isolation or isolation of a second cellular material, such as a located, different cell type or different cellular state.

The geometry of the device and wells is manufactured by any suitable technique known in the art. In some embodiments, thermal embossing, thermal deformation, and / or injection molding methods are used to generate micropatterned surfaces in plastics that are compatible with cell culture. FIG. 12 shows a diagram of the thermal embossing / thermoforming manufacturing process used herein. In some embodiments, a polystyrene film of a particular thickness (or another suitable polymer film) is placed on a heat resistant silicone support. The mold is then placed on a film with downward microposts. The entire assembly is pressed between preheated plates at 130 ° C. for 10 minutes under a load of 5N. After 10 minutes, the plate was cooled to less than 100 ° C., the micropatterned, embossed / thermoformed film was removed from the assembly and placed in a regular cell culture vessel as a 3D aggregation promoting surface. It is captured. Other temperatures, times, pressures, and materials can be within the scope of the invention.

To prevent cell adhesion, in some embodiments, the micropatterned surface is a polymer that suppresses cell adhesion, such as poly-HEMA, Pluronic, or brand name ULA (ultra-low adhesion surface) treatment. Processed using. Depending on the thickness of the initial polymer film and the processing parameters, a surface with microwells with different bottom thicknesses is produced. In some embodiments, the thickness of the polymer at the bottom of the microwell has a direct effect on oxygen permeability. The thinner bottom of the microwell allows for a better oxygen supply to the cells located inside the microwell. The fabrication method results in a surface with highly oxygen permeable microwells.

In some embodiments, the devices and systems described herein are for the movement of fluids (eg, media) and cells (eg, spheroids) in and out of various compartments, wells, etc. in such devices. Equipped with microfluidic elements. Microfluidic elements may include channels, reservoirs, valves, pumps and the like.

In vitro 3D tumor cell cultures reflect a more accurate complex in vivo microenvironment than a simple two-dimensional cell monolayer. In some embodiments, the cell culture formats with microwell patterned surfaces described herein are uniform dimensional 3D cultures compatible with routine high-throughput drug development and preclinical studies (eg,). , Causes the production of large amounts of tumor spheroids).

In some embodiments, the culture vessels described herein are induced pluripotent stem (iPS) and embryonic stem cell (ESC) cells that allow uniform and easy large-scale aggregate formation. It is used for the formation of embryoid bodies (EBs) from. In some embodiments, the medium exchange is carried out so that the EB grows continuously for several weeks. In some embodiments, the size of each aggregate is controlled because the size depends on the number of seeded cells and the culture time. In some embodiments, the aggregates are transferred to a conventional well plate that allows for larger medium volume or spheroid analysis. In some embodiments, the multi-formed EBs provide statistically important data from high-throughput analysis of transfected targets or small molecules within a single plate. The ability to support the formation of multiple 3D cell aggregates within a single culture vessel makes these vessels, such as modified Petri dishes, applicable to the selection of EB-forming clones for cell reprogramming. To do. In some embodiments, the vessels described herein also support the formation of 3D cell aggregates of various cell types associated with toxicity, such as hepatocytes and embryonic stem cells. In some embodiments, the microwell surface vessel is used, for example, for cell culture of 3D aggregates for the purpose of protein production in the field of bioprocessing engineering. In some embodiments, the cell culture formats with microwell patterned surfaces described herein allow stem cell niche co-culture, in particular single stem cell and niche co-culture in clonal cultures. Provide means. Computer identification of single stem cell and niche co-cultures can be performed in combination with established staining protocols for stem cell surface markers or stem cell differentiation markers, as well as image processing.

Micropatterned containers are also used for cell storage purposes to store cells in 3D format. In some embodiments, once the spheroids have grown to a certain size (typically in a pre-stable transient state), they are removed from the microwells and the recovered spheroids are stored frozen. , Stored for later use. Transient spheroids mean that the dimensions of the spheroids can grow continuously, while stable spheroids mean that the spheroids grow when they reach their inherent dimensional tolerances. Means to stop. Various cryopreservation methods are within the scope of the present invention, including, but not limited to, dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and the like.

Example 1: Manufacture of base material A base material according to the embodiment was produced by using the embossing method shown in FIG. FIG. 12 shows a thermal embossing / thermoforming method for microwell formation in polymer films. A hot plate 1 is provided. The hot plate was preheated to 130 ° C. An embossed mold 2 that reflects the desired well profile was provided. A layer of polymer film 3 was provided and a silicone mat 4 was provided behind the polymer film. The hot plate was heated and pressed against a polymer film supported by a silicone layer. When the hot plate was removed, a polymer film having an array of microwells with the desired well profile embossed inside was obtained.

Example 2: Cell Culture Experiments performed during the development of the embodiments described herein have, for example, a diameter (D) of about 1-3 times the desired diameter of the 3D cell aggregate, each It demonstrates the formation and culture of uniform diameter 3D cell culture aggregates in an array of separate, spatially spaced microwells with a hemispherical circular bottom and a rounded top. .. The height of the microwell (H) is equal to about 0.7-1.3 times the diameter of the bottom of the circle, and the diameter of the top opening of the microwell ( _top D) is about 1 of the diameter of the bottom of the circle. .Equal to 5 to 2.5 times.

A prototype of a T25 cell culture flask with a microwell patterned surface was produced and tested in cell culture applications to verify the uniformity of spheroid formation and retention / harvest benefits of the presented design. I conducted an experiment. FIG. 8 includes an image of a reverse replica of a microwell cross section of a T25 prototype with a circular bottom geometry. An image of spheroids formed by HT29 cells in a prototype T25 flask is shown in FIG. 10A. FIG. 10B shows a spheroid taken from a flask, which is propagated using a commercially available NUNCLON SPHERA ™ low binding surface flask, commercially available from Nunc Nunc / Thermo Fisher, shown in FIG. The superior performance of the wells described herein is comparable to that of commercial controls that have a low binding surface but no wells for defining and controlling spheroid formation. , It is reflected in the uniformity of the flask produced in the embodiment. The circular bottom geometry is a geometry that induces spheroids. During the development of the embodiments herein, experiments were performed to demonstrate the effect of microwell oxygen permeability on cell culture performance. 6-well and 12-well plates with different thickness microwells were made as described in Example 1 above. FIG. 13 shows viable cell counts measured after growing cells on a 6-well plate with a substrate with an array of microwells (described in Example 1) in microwells with different bottom thicknesses. The graph demonstrating is shown. As shown in FIG. 13, A is 70 μm thick, B is 120 μm thick, and C is 320 μm. The control was TCT-treated 1 mm thick flat polystyrene. As can be seen from the results shown in FIG. 13, thinner materials (which show more gas permeability) supported stronger cell proliferation in embodiments.

FIG. 13 shows viable cell counts measured after growing cells in 6-well plates with a substrate with an array of microwells (described in Example 1) in microwells with different bottom thicknesses. A graph demonstrating the numbers is shown. 14A and 14B show graphs comparing viable cell counts and cell proliferation rates for substrates with an array of microwells against a flat surface. FIG. 15 shows a graph showing the total titer of proteins excreted from MH677 cells cultured on a substrate having an array of microwells against a flat surface.

Culture of MH677 cells was performed for 7 or 9 days, changing medium every 2 days. The results presented in FIG. 13 demonstrate the dependence of the total viable cell count on the thickness of the bottom of the microwell. Cells cultured in oxygen permeable microwells (column A, 70 μm thick bottom) obtained an 82% higher viable cell count compared to the 320 μm thick bottom of the less oxygen permeable column C. .. Overall, cultures on microwell-patterned surfaces have higher viable cell counts (FIG. 14A) and higher proliferation per cell compared to standard flat, non-adhesive surfaces. It produces a rate (FIG. 14B) (FIG. 14). This results in an 85% higher protein yield (Fig. 15).

During the development of embodiments herein, experiments were performed using the cell culture devices and wells described herein to demonstrate the advantageous growth of cells in 3D culture over 2D culture. .. ^{Much more protein per cm 2 in} 3D culture (hm-CSF in FIG. 34A; FIG. 34B) in both CHO5 / 9 alpha cells (FIG. 34A) and BHK-21 cells (FIG. 34B) when compared to 2D. EPO) was generated.

All publications and patent documents mentioned herein and / or listed below are incorporated herein by reference. Various modifications, modifications, and variations of the features and embodiments described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the specific embodiments have been described, it should be understood that the inventions described in the claims should not be unreasonably limited to such specific embodiments. In fact, various modifications of the modes and embodiments described that are obvious to those skilled in the art are intended to be within the claims below.

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

Embodiment 1
A cell culture substrate having an array of microwells comprising at least one capillary structure and a spheroid-inducing geometry.

Embodiment 2
The at least one capillary structure is selected from the group consisting of ridges, crevices, pillars, discontinuous walls, wavy walls, mouths, paraboloids or sinusoidal wells, or combinations. The cell culture substrate according to the first embodiment.

Embodiment 3
The cell culture substrate according to the second embodiment, wherein the ridge has a circular shape, a square shape, a needle shape, or a hexagonal shape.

Embodiment 4
The cell culture substrate according to the second embodiment, wherein the fissure has a circular shape, a square shape, a needle shape, or a hexagonal shape.

Embodiment 5
The spheroid-inducing geometry includes pillars, discontinuous walls, wavy walls, rounded cell bottoms, recessed well bottoms, recesses, nib regions, discontinuous walls, or combinations. The cell culture substrate according to any one of Embodiments 1 to 4, wherein the cell culture substrate is characterized by the above.

Embodiment 6
The cell culture substrate according to any one of embodiments 1 to 5, wherein the substrate contains 2 to 10,000 microwells per square centimeter of the surface of the cell culture substrate.

Embodiment 7
The cell culture substrate according to any one of embodiments 1 to 6, wherein at least the bottom of the microwell comprises a non-adhesive surface.

8th Embodiment
The cell culture substrate according to any one of embodiments 1 to 7, wherein at least the bottom of the microwell contains a gas permeable material.

Embodiment 9
The cell culture substrate according to any one of embodiments 1 to 8, wherein the substrate contains 2 to 10,000 microwells per square centimeter of the surface of the cell culture substrate.

Embodiment 10
The cell culture substrate according to any one of embodiments 1 to 9, wherein the cell culture substrate contains at least a part of a cell culture container.

Embodiment 11
The cell culture substrate according to embodiment 10, wherein the cell culture vessel is selected from the group consisting of a multi-well plate, a dish, a flask, a tube, a multi-layer flask, a side soft flask, and a bag. ..

Embodiment 12
The cell culture substrate according to embodiment 10, wherein the cell culture vessel comprises at least two cell culture chambers separated from each other by a permeable membrane.

Embodiment 13
The cell according to any of embodiments 1-12, wherein the microwell is configured to allow fluid communication between at least one of the microwells and a single liquid reservoir. Culture substrate.

Embodiment 14
The cell culture substrate according to any one of embodiments 1 to 10, wherein the cell culture vessel comprises a mesh arranged above the microwells.

Embodiment 15
Each microwell has a top opening, a well bottom, and one or more side walls.
The top opening has an effective diameter D _top , the one or more side walls have a height H extending from the well bottom to the top opening, and the microwell is effective between the top opening and the well bottom. has a midpoint D _intermediate diameter, D _top: D ratio of the _intermediate is 1.5: 2.5, characterized in that a cell culture substrate according to any of embodiments 1 to 14.

Embodiment 16
The cell culture substrate according to embodiment 15, wherein the _{upper part of} D _{is in the middle} of 1.5 to 2.5D.

Embodiment 17
The cell culture substrate according to embodiment 15, characterized in that H = 0.7 to 1.3D _{intermediate.}

Embodiment 18
The cell culture substrate according to embodiment 15, wherein the _middle D is 200 to 1000 μm.

Embodiment 19
The step of filling the cell culture vessel according to any one of embodiments 10 to 12 with a culture medium;
A method for culturing spheroids, which comprises the step of adding spheroid-forming cells to the culture medium.

20th embodiment
19. The method of embodiment 19, further comprising the step of replacing or exchanging the culture medium.

References

1. H. Dolznig, A. Walzl, Organotypic spheroid cultures to study tumor- stroma interactions during cancer development. Drug discovery today, V 8., No 2-3, 2011, 113-118
2. J. Engelberg, G. Ropella, Essential operating principles for tumor spheroid grwth. BMC Systems Biology 2008, 2, 110
3. Y. Tung, A. Hsiao, S. Alen. High-throughput 3D spheroid culture and drug testing using 384 hanging drop array. Analyst, 2011, 136, 473-478
4. J. Friedrich, C. Seidel, R. Ebner. Spheroid -based drug screen: considerations and practical approach. Nature protocols, 2009, vol4 no3, 309-323
5. F. Hirschhaeuser, H. Menne, C. Dittfeld, Multicellular tumor spheroids: An underestimated tool is catching up again. Journal of Biotechnology, 2010, 148, 3-15
6. T. Bartosh, J. Ylostalo, A. Mohammadipoor. Aggregation of human mesenchymal stromal cells into 3D spheroids enhances their anti-inflammatory properties. PNAS, 2010, 107, no 31, 13724-13729
7. J. Frith, M. Res, B. Thomson. Dynamic three-dimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential. Tissue engineering, 2010, 16, no 4, 735-749
8. S. Sart, A.Tsai, Y. Li. Three-dimensional aggregates of mesenchymal stem cells: cellular mechanisms, biological properties and applications. Tissue engineering, 2013, Part B, 00, no 00, 1-16

100-well array 110 Mouth 111 Top opening / Top Mouth 112 Mouth bottom 113 Side wall 115 Well 116 Well bottom 170 Ridge 270 Crack 401 Microwell 402 Frame 403 Side wall 500 Spheroid 501 Pillar 505 Space 510 Top 515 Well 800 Flask 815 Top Top 820 Side Wall 850 Cell Culture Chamber 860 Port 861 Screw Lid 1110 Base Material 1114 Base Material 1300 Cell Culture Medium 1400 Cell Culture Equipment 1410A-C Cell Culture Compartment 1415 Tray 1420 Aeration Manifold 1422 Cap 1425 Opening 1426 Opening 1426 1429 Ventilation column 1430 Filled manifold 1432 Cap 1435 Opening 1439 Row 1440 Side wall 1441 Upper space 1442 Ventilation opening 1443 Filled opening 1450 Upper inner surface / Upper surface 1460A-C Passage / Conduit space 1500 Spacer 1510 Plate 1601 Hexagonal well 2100 Cell culture device Base material 2115 Well 2120 Side wall 2152 Culture chamber 2154 Second chamber 2162 First port 2164 Second port 2202 Spheroid cell 3100 Cell culture device 3110 Base material 3111 Plate 3113 Frame 3115 Well 3116 Pen tip area 3118 Top opening 3120 Side wall surface 3121 Variation points 3122 Lower side wall surface 3123 points 3124 Upper side wall surface 3130 Spheroid 3140 Cell culture volume 3142 Height 3144 Diameter dimension 3145 Second volume 3150 Base material 3600 Cell culture assembly 3610 Bottom plate 3611 Main surface 3615 Microwell / well 3620 Side wall 3650 Cell culture device 3670 Mesh 4100 Cell culture device 4110 Bottom plate 4115 Microwell / well 4120 Side wall 4140 Entrance 4145 Exit 4200 Cell culture assembly 4560 Frame 4570 mesh

Claims (10)

A cell culture substrate containing an array of microwells, each microwell into said microwell defined by an upper well edge, a corrugated microwell side wall, and a rounded well bottom. With an opening,
A cell culture substrate in which a plurality of the wavy side walls are aligned so that a plurality of the microwells are generated in a gap between the plurality of the wavy side walls. The cell culture substrate according to claim 1, wherein at least the rounded well bottom comprises a non-adhesive surface and the rounded well bottom comprises a gas permeable material. The cell culture substrate according to claim 1 or 2, wherein the cell culture substrate contains at least a part of a cell culture container. The cell culture substrate according to claim 3, wherein the cell culture vessel is selected from the group consisting of a multi-well plate, a dish, a flask, a tube, a multi-layer flask, a side soft flask, and a bag. The cell culture substrate of claim 1, wherein the microwells are configured to allow fluid communication between at least one of the microwells and a single liquid reservoir. The cell culture substrate according to claim 1, wherein the side wall of the microwell extends from the opening of the microwell to the bottom of the microwell in a corrugated manner. The cell culture substrate according to claim 1, wherein each lower portion of the microwell extends below the side wall of the corrugated microwell to form a circular well bottom. The cell culture substrate according to claim 1, wherein the side wall of the microwell has a wavy shape by having a region in which the plurality of side walls are periodically separated from each other and then approached. The periodic region of the side wall is a region in which the side walls are far apart and then approach without contacting each other, and rows of microwells are formed in the gaps between the wavy side walls. The cell culture substrate according to claim 8. The periodic region of the side wall is a region in which the side walls are far apart and then approach each other so as to come into contact with each other, and a row of microwells is formed in the gap between the wavy side walls. The cell culture substrate according to claim 8.

Images