KR102527308B1 - Devices and Methods For Generation and Culture of 3D Cell Aggregates - Google Patents

Abstract

The present disclosure relates to devices, systems and methods for culturing cells. In particular, devices and methods for generating and culturing three-dimensional cell aggregates are provided.

Description

Devices and methods for generation and culture of 3D cell aggregates {Devices and Methods For Generation and Culture of 3D Cell Aggregates}

This application claims the benefit of U.S. Provisional Patent Application No. 62/072015, filed on October 29, 2014; 62/072103 filed on October 29, 2014; 62/072088 filed on October 29, 2014; claiming priority to serial number 62/094471 filed on 19 December 2014; The entire contents of these are incorporated herein by reference.

The present disclosure relates to devices, systems and methods for culturing cells. In particular, devices and methods for generating and culturing three-dimensional cell aggregates are provided.

Three-dimensional (3D) cell culture is the growth of cells in an artificially-created environment that allows cells to grow and/or interact with each other in three dimensions. 3D cell culture represents an improvement over methods of growing cells in 2D (eg, Petri dishes), at least because 3D conditions more accurately model the in vivo environment.

Cells cultured in three dimensions, such as spheroids, may exhibit functionality that is rather close to living organisms compared to controls cultured in two dimensions as a monolayer. In a two-dimensional cell culture system, cells can be attached to the substrate on which they are cultured. However, when cells grow in three dimensions, such as spheroids, they interact with each other rather than attach to the substrate. One problem with spheroid-based assays is that assay results typically depend on the size of the spheroid. Variations in parameters, such as, for example, seeding density from system to system and growth time, can affect assay reproducibility from system to system or well to well within a given system. Thus, maintaining constant spheroid size between spheroids grown in individual wells can present challenges.

As the density of cells grown in a cell culture device increases, a larger volume of cell culture medium or more frequent exchange of cell culture medium may be required to maintain the cells. However, the increased frequency of media exchanges can be inconvenient. Additionally, an increased volume of cell culture medium may lead to an undesirably increased height of the medium over the cultured cells. As medium height increases, the rate of gas exchange of cells through the medium decreases.

Cells grow densely as spheroid clusters in wave bags, spinners and shakers. However, the size of spheroids grown in these devices is not constant, and the shear inherent in these devices tends to break the spheroids into smaller clusters. Moreover, such devices may not be able to achieve cell densities high enough to meet current needs.

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

Cells cultured in three dimensions, such as spheroids, can exhibit functionality rather close to in vivo compared to controls cultured in two dimensions as a monolayer. In a two-dimensional cell culture system, cells can be attached to the substrate on which they are cultured. However, when cells grow in three dimensions, such as spheroids, the cells interact with each other rather than adhere to the substrate. Cells cultured in three dimensions more closely resemble in vivo tissues in terms of cell-to-cell communication and development of extracellular matrices. Thus, spheroids provide an excellent model for cell migration, differentiation, survival and growth, and thus a better system for research, diagnosis, and testing of drug efficacy, pharmacology, and toxicity.

In some embodiments, a substrate comprising or containing microwells or an array of wells is provided. The substrate may form part of a cell culture apparatus or device. For example, the substrate may form part of a multiwell plate, flask, dish, tube, multi-layer cell culture flask, bioreactor, or other laboratory vessel intended to grow cells or spheroids. there is. Microwells or wells (the terms "microwell" and "well" are used interchangeably in this disclosure) are constructed and arranged to provide an environment conducive to the formation of spheroids in culture. That is, in an embodiment, the microwell has a spheroid-inducing geometry. Additionally, the well is structured and arranged to provide for the movement of liquid into and out of the well without entrapment of air between the liquid or liquid droplets and the substrate introduced into the well. That is, in an embodiment, the microwell has a capillary structure. For example, the well in which the cells are grown may be non-adherent to the cells such that the cells within the well associate with each other to form spheres. The spheroid expands to the size limit imposed by the geometry of the well. In some embodiments, the wells are coated with an ultra-low binding material to make the wells non-adherent to the cells. In some embodiments, the substrate may include 2-10000 microwells per cm 2 of the surface of the cell culture substrate.

In some embodiments, a cell culture device has a frame comprising a footprint of the device, the substrate of which is configured such that cells cultured in the device form spheroids. For example, cell culture substrates within the device are non-adherent to the cells such that the cells bond to each other instead of the substrate. The cell culture substrate is further composed of a plurality of microwells (or wells), the geometry of which allows cells grown in the wells to form similar-sized cell aggregates or spheroids. The spheroid expands to the size limit imposed by the geometry of the microwell. In some embodiments, the wells are subjected to a low-binding treatment or coated with an ultra-low binding material to make the wells non-adherent to cells.

Examples of non-adhesive materials include perfluorinated polymers, olefins, or similar polymers or mixtures thereof. Other examples include agarose, non-ionic hydrogels such as polyacrylamide, polyethers such as polyethylene oxide and polyols such as polyvinyl alcohol, or similar materials or mixtures thereof. For example, a combination of non-adherent wells, well geometry (eg, size and shape), and/or gravity induces cells cultured in the well to self-assemble into spheroids. . Some spheroids retain differentiated cellular functions that, compared to cells grown in monolayers, respond rather closely to in vivo. Other cell types, such as mesenchymal stromal cells, retain their pluripotency when cultured as spheroids.

In some embodiments, the systems, devices and methods herein include one or more cells. In some embodiments, the cells are cryopreserved. In some embodiments, the cells are in 3-dimensional culture. In some such embodiments, the systems, devices and methods include one or more spheroids. In some embodiments, one or more cells are actively dividing. In some embodiments, systems, devices, and methods may include culture media (e.g., nutrients (e.g., proteins, peptides, amino acids), energy (e.g., carbohydrates), essential metals and minerals (e.g., calcium, magnesium, iron, phosphates, sulfates), buffers (e.g. phosphates, acetates), indicators for pH change (e.g. phenol red, bromo-cresol purple), optional agents (e.g. chemical agents, antibacterial agents), etc.). In some embodiments, one or more test compounds (eg, drugs) are included in systems, devices, and methods.

Cells of various types can be cultured. In some embodiments, the spheroid contains a single cell type. In some embodiments, spheroids contain more than one cell type. In some embodiments where more than one spheroid is grown, each spheroid is of the same type, but in other embodiments, two or more spheroids of different types are grown. Cells grown in spheroids can be natural cells or altered cells (eg, cells containing one or more non-naturally occurring genetic alterations). In some embodiments, the cell is a somatic cell. In some embodiments, the cell is a stem cell or progenitor cell (e.g., pluripotent, multi-potent, fate determined, immortalized, etc.) in any desired state of differentiation. For example, embryonic stem cells, induced pluripotent stem cells). In some embodiments, the cell is a disease cell or disease model cell. For example, in some embodiments, the spheroid comprises one or more types of cancer cells or cells that can be induced to a hyper-proliferative state (eg, transformed cells). Cells include adrenal, bladder, blood vessels, bone, bone marrow, brain, cartilage, cervix, cornea, endometrium, esophagus, gastrointestinal tract, immune system (e.g., T lymphocytes, B lymphocytes, white blood cells, macrophages and dendritic cells), Liver, lungs, lymph glands, muscles (eg heart muscle), nerves, ovaries, pancreas (eg islet cells), pituitary gland, prostate, kidneys, saliva, skin, tendons, testes and thyroid, but hereby It can be derived from or formed from any desired tissue or organ type, including but not limited to. In some embodiments, the cell is a mammalian cell (eg, human, mouse, rat, rabbit, dog, cat, cow, pig, chicken, goat, horse, etc.).

Cultured cells are used in a variety of research, diagnostics, drug screens and tests, therapeutics, and industrial applications.

In some embodiments, cells are used for the production of proteins or viruses. Systems, devices and methods for culturing large numbers of spheroids in parallel are particularly effective for protein production. Three-dimensional culture allows for increased cell density and higher protein yield per square centimeter of cell growth surface area. Any desired protein or virus for vaccine production can be grown in cells and isolated or purified for use as desired. In some embodiments, the protein is a native protein to the cell. In some embodiments, the protein is a denatured-protein. In some embodiments, the protein is recombinantly expressed. Preferably, the protein is overexpressed using a non-native promoter. Proteins can be expressed as fusion proteins. In some embodiments, a purification or detection tag is expressed as a fusion partner to a protein of interest to facilitate its purification and/or detection. In some embodiments, fusions are expressed with a cleavable linker to allow isolation of the fusion partner after purification.

In some embodiments, the protein is a therapeutic protein. Such proteins may replace deficient or abnormal proteins (eg insulin), increase existing pathways (eg inhibitors or agonists), provide new functions or activities, or modify molecules or organisms. proteins and peptides that interfere with, or deliver other compounds or proteins (eg, radionuclides, cytotoxic drugs, effector proteins, etc.). In some embodiments, the protein is an immunoglobulin, such as an antibody (eg, monoclonal antibody) of any type (eg, humanized, bi-specific, multi-specific, etc.). Therapeutic protein categories include antibody-based drugs, Fc fusion proteins, anticoagulants, antigens, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, and interferons. , interleukins, and thrombolytics, but are not limited thereto. Therapeutic proteins can be used to prevent or treat cancer, immune disorders, metabolic disorders, genetic disorders, infections, 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 expresses a detectable moiety (eg, a fluorescent moiety, a luminescent moiety (eg, luciferase), a colorimetric moiety, etc.) do.

In some embodiments, the protein is an industrial protein. Industrial proteins include, but are not limited to, food ingredients, industrial enzymes, agricultural proteins, assay enzymes, and the like.

In some embodiments, the cells are used for drug discovery, characterization, efficacy testing, and toxicity testing. These tests include pharmacological effect evaluation, carcinogenicity evaluation, medical imaging agent characterization, half-life evaluation, radiation safety evaluation, genotoxicity testing, immunotoxicity testing, reproduction and developmental testing, drug interaction evaluation, dose evaluation, adsorption evaluation, and predisposition. Disposition assessments, metabolic assessments, clearance studies, and the like, but are not limited thereto. Certain cell types may be subjected to special tests (e.g., hepatocytes for liver toxicity, renal proximal tubular epithelial cells for nephrotoxicity, vascular endothelial cells for vascular toxicity, neuronal and glial cells for neurotoxicity, cardiomyocytes for cardiotoxicity, skeletal muscle cells for rhabdomyolysis, etc.). Treated cells can have any number of changes, including but not limited to membrane integrity, cellular metabolite content, mitochondrial function, lysosomal function, apoptosis, genetic alterations, gene expression differences, and the like. It can be evaluated for any desired parameters.

In some embodiments, the cell culture device is a component of a larger system. In some embodiments, the system can generate multiple (e.g., 2, 3, 4, 5, ..., 10, ..., 20, ..., 50, ..., 100, ..., 1000, etc.) including these cell culture devices. In some embodiments, the system includes an incubator to maintain the culture device at optimal culture conditions (eg, temperature, atmosphere, humidity, etc.). In some embodiments, the system includes a detector for imaging or otherwise analyzing cells. These detectors include fluorometers, luminometers, cameras, microscopes, plate readers (eg, PERKIN ELMER ENVISION plate readers, PERKIN ELMER VIEWLUX plate readers), cell analyzers (GE IN Cell Analyzer 2000 and 2200, THERMO/CELLOMICS CELLNSIGHT High Content Screening Platform) and confocal imaging systems (eg, PERKIN ELMER OPERAPHENIX High Efficiency Content Screening System, GE INCELL 6000 Cell Imaging System). In some embodiments, the system includes perfusion systems or other components for supplying, re-supplying, and circulating culture medium or other components to the cultured cells. In some embodiments, the system includes robotic components (eg, pipettes, arms, plate movers, etc.) for automating handling, use, and/or analysis of the culture device.

In the handling of microwell format microplates or other containers with microwells, care must be taken to ensure complete displacement of air from the microwells upon introduction of aqueous liquids, particularly during addition of liquids to the microwells. When a liquid is added to a vessel containing a well, air may be trapped beneath the liquid but within the well (eg, a microwell), especially if the well has a circular cross-section. The surface tension of the aqueous liquid added to the well is strong, so the droplet tends to remain spherical. A drop of sphere can easily plug a circular hole of similar size, resulting in air being trapped within the hole (eg, microwell).

In some embodiments, provided herein are well geometries that reduce the likelihood of air being entrapped in the microwells while maintaining the cell culture properties of the wells (eg usefulness in 3D cell culture). In some embodiments, the well geometry (eg, microwell geometry) allows efficient displacement of air upon introduction of liquid into the wells. In some embodiments, the well geometry provides pathways for the flow of liquid into the wells without blocking the evacuation of air from the wells. In some embodiments, the well geometry provides pathways for trapped air to escape. In some embodiments, various well geometries are provided herein that promote air displacement in microwells and allow entry of liquid into microwells while maintaining confinement dimensions for cell aggregation.

In an embodiment, the present disclosure provides a device (e.g., a multiwell plate, petri dish, flask, multi-layer flask, or hyperstack (e.g., a multi-well plate, petri dish, or hyperstack) for culturing and analyzing spheroid cell masses or other aggregate cell colonies. HyperStacks)). In some embodiments, a device includes at least one chamber (eg, wells (eg, wells (eg, For example, macrowells), flasks, etc.). In some embodiments, the geometry of the aperture, sidewall(s), and bottom may be: (1) reagents (eg, displacement of air from the chamber (e.g., without air trapped in or below the liquid) upon dispensing (e.g., a liquid reagent) into the microwells, which reduces the possibility of entrapment of air below the surface of the liquid in the microwells. one or more (e.g., all) of a path for flow of liquid, (3) a path for escape of air upon introduction of liquid into the microwell, and/or (4) a path for escape of air trapped below the surface of the liquid. make it possible

In some embodiments, a device having a surface comprising wells (eg, microwells) and one or more wells (eg, microwells) in which the surface does not have any polygonal angle (eg, 90 degrees). is prepared

In some embodiments, the wells (eg, microwells) have a cross-sectional shape that approximates a sine wave. In this embodiment, the bottom of the well is round (eg, rounded hemispherical), the sidewalls increase in diameter from the bottom to the top of the well, and the boundaries between the wells are rounded. As such, the top of the well is not finished at right angles. In some embodiments, the well has a diameter midway between the bottom and top (D) (also referred to as D _{half - way} ), a diameter at the top of the well (D _top ), and a height from the bottom to the top of the well (H ) has In these embodiments, D _top exceeds D. Both the relative and absolute dimensions of the wells can be selected for desired culture conditions. For spheroid growth, the diameter (D) is preferably 1 to 3 times the desired diameter of the 3D cell aggregates to be cultured in the well. The height (H) is 0.7 to 1.3 times D. The diameter (D _top ) is 1.5 to 2.5 times D. D is preferably (e.g. 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800 or 2000 micrometers, and 100 micrometers (μm) to about 2000 are micrometers. However, optional 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 therebetween (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, etc.). D is from 100 μm to 10,000 μm or any value therebetween (eg 100, 200, 500, 1000, 2000, 5000) or a range therebetween (eg 100-2000, 200-1000, 300 -700, 400-600, 500, etc.). H is 0.5 to 10 times D (e.g., 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 between can be any value or range therein). D _top is 1.1 to 5 times D (eg, 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 therebetween). can

The barriers between adjacent wells (eg, microwells) may have the opposite shape of the neighboring wells, may have a larger or smaller diameter (D _B ), or may be of a different shape ( For example, the shape of the well-bottom may be different from the shape of the well/barrier top, see eg FIG. 2). To maximize the number of wells in a given surface, D _B is preferably smaller than D. D _B is 1.1 times to 5 times greater than D (eg, 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 therebetween). may be larger or smaller.

In certain embodiments, the cell culture device herein comprises a plurality of wells, structures that cause cells cultured in the wells to form spheroids of a particular diameter. A cell culture device may include a structure defining a plurality of wells. In some embodiments, each of the plurality of wells defines an upper opening, a lower well, and a sidewall surface extending from the upper opening to the lower well. The sidewall surface defines a pen tip area between the upper aperture and the well-bottom. The cell culture volume is defined by the bottom surface, part of the side wall surface, and the pen tip area. In some embodiments, the pen tip zone is between 50 micrometers and 700 micrometers (e.g., 50 μm, 75 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm) from the bottom of the well. and any range therebetween) at a height ranging from 100 micrometers to 700 micrometers (e.g., 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm and any range therebetween). bounded by the diameter of the range. The side wall surface or cell culture volume is dimensioned to control the size of the spheroids growing in each well. The pen tip region is a spheroid derived geometry.

Embodiments include, for example, no air bubble entrapment during cell seeding or media exchange, high retention of 3D cell aggregates during media exchange, ease of harvesting spheroids from large surface areas, gas permeability, media storage over multiple wells, It offers a number of features including spheroid-confining wells, and/or the ability to generate large and uniformly sized spheroids.

In some embodiments, a well described herein extends into the well from an upper opening, which provides a path for air to escape upon entry of liquid into the well, and extends from the upper opening to the bottom of the well, or from the upper opening to the bottom of the mouse. may include one or more capillary structures (eg, ridges, fissures, corners, acute angles, corrugations, pillars, etc.). Suitable well geometries within the scope of the embodiments described herein include: (a) a square cross-section top opening, a round (eg, concave) well-bottom, and a well-bottom from a square cross-section at the top of the well. wells with sidewalls transitioning to a circular cross section; (b) a well having one or more protruding ridgelines extending from an upper opening (eg, a circular cross-section upper opening) to a well-bottom (eg, a round (eg, concave) well-bottom); eg, see FIG. 1B); (c) a well (e.g., having one or more clefts extending from an upper opening (e.g., a circular cross-section upper opening) to a well-lower (e.g., a round (eg, concave) well-bottom). , see Fig. 2b); (d) a lower portion of the well having a rounded bottom and an upper portion defined by first and second sidewalls that do not completely surround the well, and wherein the well has a sidewall bordering the well (e.g., FIG. 27 or Figure 28); (e) A well in which at least one sidewall has a convex cross-section, thereby creating an acute angle between two sidewalls that may be formed by a column (see FIG. 5). In some embodiments, the wells include variations and/or combinations of the above geometries.

In an embodiment, a method of manufacturing a cell culture device comprising a well described herein is provided.

In embodiments, provided herein are methods of using a cell culture device comprising the wells described herein, eg, in spheroid cell culture or cellular assays.

In some embodiments, provided herein is a cell culture device comprising a frame having wells disposed therein, wherein the wells include: (a) a top opening; (b) a well-bottom with a round cross-sectional geometry; (c) a sidewall or sidewalls extending from the well-bottom to the upper opening; (d) optionally a mouse; and (e) optionally a capillary structure that facilitates the introduction of liquid into the interior of the well and the evacuation of air from the well without the ever-repeated formation of pockets of air beneath the surface of the liquid.

In some embodiments, the well-bottom has a circular cross-sectional geometry, wherein the upper opening has a polygonal cross-sectional geometry, and wherein the sidewall transitions from a circular to a polygonal cross-sectional geometry, thereby introducing liquid between the sidewalls into the well. and a corner, which serves as a capillary structure that facilitates the evacuation of air from the well. In some embodiments, the transition of the sidewall cross-sectional geometry does not provide an obstruction to fluid flow into or out of the well. In some embodiments, the upper opening has a square or hexagonal cross-sectional geometry.

In some embodiments, the structural feature that facilitates the introduction of liquid into and discharge of air from the well is a sidewall or a ridge protruding from the sidewall and extending from the well-bottom to the upper opening. In some embodiments, the well extends from the well-bottom to the upper opening and protrudes from, or substantially such a sidewall or sidewalls, 1 to 20 (eg, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) ridges. In some embodiments, the well-bottom has a circular cross-sectional geometry and the upper opening has a circular or polygonal cross-sectional geometry. 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 span the entire distance from the upper opening to the well-bottom.

In some embodiments, the structural feature that facilitates the introduction of liquid into the well and the discharge of air from the well is a slit in the sidewall or sidewalls extending from the well-bottom to the upper opening. In some embodiments, the well extends from the bottom of the well to the top opening and there are 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). In some embodiments, the well-bottom has a circular cross-sectional geometry and the upper opening has a circular or polygonal cross-sectional geometry. 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 span the entire distance from the upper opening to the well-bottom.

In some embodiments, a well is defined by three or more adjacent pillars (eg, 3, 4, 5, 6, 7, 8, 9, 10), a portion of a side of each pillar being a sidewall of the well. form; and wherein the limited space between adjacent columns forms a structural feature that facilitates the introduction of liquid into and the discharge of air from the well. In some embodiments, the well-bottom has a circular cross-sectional geometry and extends down the column.

In some embodiments, provided herein is a cell culture device comprising a frame comprising a plurality of wells therein; wherein the wells are arranged in at least one row; wherein the row is defined by two corrugated sidewalls aligned such that a gap between the sidewalls widens and narrows with each corrugation; Here, the upper portion of each well is bounded by a widened gap between two narrow gaps in the sidewall, so that the upper portions of adjacent wells are in fluid communication; and wherein the lower portion of each well extends below the corrugated sidewall and forms a circular well-bottom. In some embodiments, a device includes multiple rows of wells.

In some embodiments, the sidewall, sidewalls, and/or bottom-well are gas permeable and liquid impermeable. That is, in some embodiments, the substrate on which the wells are formed is gas permeable and liquid impermeable.

In some embodiments, the sidewall or sidewalls are opaque and the well-bottom is transparent.

In some embodiments, the bottom-well includes a concave arcuate surface.

In some embodiments, the sidewall, sidewalls, and/or well-bottom comprises a low-adhesion or non-adhesion material and/or is coated with a low-adhesion or non-adhesion material.

In some embodiments, the cell culture device contains 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 therein).

In some embodiments, surfaces with microwell patterns are incorporated into a wide range of cell culture products. In some embodiments, the bottom of a well (eg, a macrowell) is patterned with a microwell surface, for example in 12 well, 24 well and 6 well plates. In some embodiments, microwell surfaces are incorporated into large surface area cell culture vessels, such as T25, T75, T125, T175 and T250 flasks and products of the CellSTACK and HYPERStack lines. In some embodiments, cell culture in a large surface area vessel having a large number of the microwells produces a large amount of 3D cell aggregates amenable to cell therapy applications, clonogenic culture, stem cell niches or niche cell co-cultures. yields

In some embodiments, a cell culture device herein includes a lower plate defining a major surface, one or more sidewalls extending from the lower plate defining a reservoir, and a plurality of wells formed in the major surface. Each well defines an upper opening that is flush with the major surface and opens the reservoir, and a well-lower nadir located below the major surface. In contrast to conventional well plates, the plates described herein define a reservoir above the surface of the well, which allows for an increased volume of cell culture medium to be used, providing less frequent medium exchange. See eg FIG. 28 .

In various embodiments, a cell culture device having one or more cell culture compartments is described. In some embodiments, the cell culture compartments are stacked. In some embodiments, each cell culture compartment includes a substrate defining a structured surface defining a plurality of gas permeable wells. In some embodiments, the well is in gas communication with the exterior of the device directly, through a gas permeable material, or through an outlet or tracheal space. In some embodiments, a cell culture device is described wherein the wells have, at least in part, a substrate with an array of microwells made of a gas permeable material. Thus, in some embodiments, the device is used to culture cells in wells while having a height of cell culture medium above the cultured cells that may be too high in conventional cell culture devices for efficient metabolic gas exchange. As the cells are cultured in gas permeable wells in gas communication with the outside of the device, gas exchange occurs through the well to overcome the lack of gas exchange through the cell culture medium due to the height of the medium above the cells.

In certain embodiments, a cell culture device includes one or more cell culture compartments. In some embodiments, each cell culture compartment comprises a substrate having an interior and having a first major surface and an opposing second major surface; The first major surface defines a structured surface on the interior of the compartment. In some embodiments, the structured surface defines a plurality of gas permeable wells. In some embodiments, the well is in gas communication with the exterior of the device.

In some embodiments, filling a cell culture device described herein with a culture medium; and adding the spheroid-forming cells to a culture medium. In some embodiments, the method further comprises replacing/changing the medium (eg, daily, continuously, etc.).

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

Additional features and advantages of the subject matter of the present disclosure will be set forth in the following detailed description, and in part will be apparent to those skilled in the art from the detailed description, including the following detailed description, claims as well as the appended drawings, It will be appreciated by practicing the subject matter of this disclosure as described herein.

It will be understood that the foregoing background and the following detailed description are intended to present specific examples of the subject matter of the present disclosure, and to provide an overview or framework for understanding the nature and nature of the subject matter of the present disclosure as claimed. The accompanying drawings are included to provide a further understanding of the subject matter of the present disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate various embodiments of the subject matter of the present disclosure and, together with the detailed description, serve to explain the principles and operation of the subject matter of the present disclosure. Additionally, the drawings and detailed description are meant to be illustrative only and in no way limit the scope of the claims.

1A and 1B are schematic diagrams of a representative embodiment of an array 100 of wells. 1A is an example of a cross section. FIG. 1B is a plan view of a representative embodiment of an array of wells, taken at line BB in FIG. 1A.
2A and B are schematic diagrams of another representative embodiment of an array 100 of wells. 2A is an example of a cross section. FIG. 2B is a plan view of a representative embodiment of an array of wells, taken at line BB in FIG. 2A.
3A-D are schematic diagrams of another representative embodiment of an array 100 of wells. 3A is an example of a cross section. FIG. 3B is a top view of a representative embodiment of an array of wells, taken at line BB in FIG. 3A. 3C is a diagram of an array of wells having a sinusoidal or parabolic shape. 3D is a side view of an array of wells containing spheroids, in an embodiment.
4A-D are schematic diagrams showing another representative embodiment of a spheroid-containing cell culture compartment, or array of wells, in the corrugated embodiment. 4A and 4C are top views of a corrugated embodiment of a substrate having an array of wells, and FIGS. 4B and 4D are partial cut-away views of the same embodiment.
5A-E are schematic diagrams of additional representative embodiments in which a series of posts define wells. 5A-B are plan views, and FIGS. 5C-5E are partially cut-away perspective views of representative embodiments.
6a-e show representative, non-limiting examples of cross-sectional geometries for ridges protruding from the sidewalls.
Figures 7a-e show representative non-limiting examples of cross-sectional geometries for crevices in sidewalls.
8 shows a culture flask with a bottom surface micropatterned with an array of microwells.
FIG. 9 shows an enlarged view of a substrate, micropatterned with an array of microwells, forming the lower surface of the flask shown in FIG. 8 .
FIG. 10A shows spheroids in HT29 cells inside microwells of a micropatterned T25 spheroid forming flask, as shown in FIG. 9 . 10B shows spheroids harvested from micropatterned T25 spheroid forming flasks.
11A and B show micrographs of spheroids or 3D aggregates formed on NUNCLON SPHERA™ low bonding surfaces available from Nunc/ThermoFisher. 11A shows human ESC cells, and FIG. 11B shows murine ESC cells.
12 is an illustration of a method of fabricating a substrate having a multiwell array, according to an embodiment.
13 shows a graph showing the number of viable cells measured after growing cells in a 6 well plate having a substrate with an array of microwells (as described in Example 1), in microwells with different bottom thicknesses. .
14A and 14B show graphs comparing viable cell count (FIG. 14A) and cell productivity (FIG. 14B) for a substrate with an array of microwells compared to a flat surface.
15 presents a graph depicting total protein titer secreted from MH677 cells cultured on a substrate with an array of microwells compared to a flat surface.
16 is an image of an embodiment of a structured surface.
17 is a photograph of cells grown in the wells of an embodiment of the structured surface.
18 is a side view illustrating a specific example of a cell culture device including a porous membrane support.
19 is a side view illustrating an additional embodiment of a cell culture device comprising a porous membrane support.
20 is a side view illustrating an additional embodiment of a cell culture device comprising a porous membrane support illustrating co-cultivation of cells.
21 is a schematic perspective view of a specific example of a cell culture device.
22 is a schematic cross-sectional view of an embodiment of a cell culture device.
23 is a schematic cross-sectional view of an embodiment of a cell culture device.
24A is a schematic bottom view of an embodiment of a tray that can be used to form part of an apparatus as shown in any one of FIGS. 21-23.
24B is a schematic perspective view of an embodiment of the tray shown in FIG. 24A.
25 is a schematic side view of an embodiment of a cell culture device.
26 is a perspective view of a specific example of a cell culture device having wells.
27 is a schematic cross-sectional view of an embodiment of a plurality of wells.
28 is a schematic cross-sectional view of an embodiment of a plurality of wells.
29 is an enlarged schematic cross-sectional view of an embodiment of a plurality of wells.
30 is a schematic perspective view of an embodiment of a cell culture device having plates and wells.
31 is a schematic perspective view of an embodiment of a cell culture device having plates and wells.
32 is a schematic perspective view of an embodiment of a cell culture device and an insert comprising a mesh.
33 is a schematic perspective view of an embodiment of a device having an inlet and an outlet.
34A and 34B are graphs showing protein production per cm 2 of a 96-well spheroid micro-well plate in (A) CHO 5/9 alpha cells and (B) BHK-21 pc.DNA3-1HC cells. indicate

Various embodiments of the present disclosure will be described in detail with reference to the drawings. Reference to various embodiments does not limit the scope of the invention. Additionally, any embodiments described herein are not limiting and merely describe some of the many possible embodiments of the claimed invention. Like numbers used in the drawings refer to like elements, steps and the like. However, it will be understood that the use of numbers to refer to elements in a given figure is not intended to limit elements in other figures labeled with the same number. Additionally, the use of different numbers to indicate elements is not intended to indicate that different numbered elements may not be identical or dissimilar 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 understanding of certain terms used frequently herein and are not intended to limit the scope of the present disclosure.

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

"Includes", "comprising" or similar terms are meant to be inclusive, but not exclusive and inclusive.

For example, amounts, concentrations, volumes, process temperatures, process times, yields, flow rates, pressures, viscosities, and similar values, and ranges thereof, of components in a composition, used to describe embodiments of the present disclosure; or "about" altering the dimensions of a component, and similar values and ranges thereof, is used, for example, to prepare a substance, composition, composite, concentrate, component part, product of manufacture, or formulation for use. through normal measuring and handling procedures; through careless mistakes in this procedure; through differences in the manufacture, source, or purity of the starting materials or components used to carry out the methods; and through similar considerations, changes in numerical quantities that can occur. The term “about” also encompasses amounts that differ due to aging of the composition or formulation having a specific initial concentration or mixture, and mixing or processing of the composition or formulation having a specific initial concentration or mixture.

"Optional" or "optionally" means that the steps, features, conditions, properties, or structures described in Naju occur/exist or do not occur/exist, but are still within the scope described.

The words "preferred" and "preferably" refer to embodiments of the present disclosure that may provide particular benefits under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude the other embodiments from the scope of the present disclosure.

The devices, methods of making the devices, and methods of using the devices described herein may include components or steps described herein, and may additionally include other components or steps not described herein.

As used in this specification and the appended claims, the term "or" is generally used in its sense including "and/or" unless the content clearly dictates otherwise. The term "and/or" means one or all of the listed elements or a combination of two or more of the listed elements.

As used herein, "having", "having", "having", "comprising", "comprising", "comprising", "including", "comprises", or similar terms , is used in an open and inclusive sense and generally means "including but not limited to", "including but not limited to" or "including but not limited to".

Ranges may be expressed herein from “about” one particular value and/or from “about” another particular value. Where such ranges are indicated, embodiments include one particular value and/or another particular value. Similarly, when values are expressed as approximate values, by use of the antecedent "about" it will be understood that the particular value forms another aspect. It will further be understood that the endpoints of each of the ranges have meaning in relation to the other endpoints and independently of the other endpoints.

As used herein, the “a,” “a,” and “the singular” include plural referents unless the context dictates otherwise.

Abbreviations well known to those skilled in the art may be used (e.g., "h" or "hrs" for hour or hours, "g" or "gm" for grams, "mL" for milliliters, " rt", "nm" and similar abbreviations for nanometers).

Specific and preferred values disclosed for components, components, additives, dimensions, conditions and like aspects, and ranges thereof, are illustrative only; Unless otherwise specified, other limited values or other values within the limited range are not excluded. The devices and methods of this disclosure include any value or any combination, including the values set forth herein, specific, more specific and preferred values, and intermediate values and ranges therebetween, either explicit or implied. do.

Also herein, recitation of numerical ranges by endpoints includes all numbers subsumed within that range. (eg 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). If the range of values is a specific value such as "greater than" or "less than", the value is included in the range.

Any directions, such as "up", "down", "left", "right", "upper", "lower", "upper", "lower" and other directions and orientations herein, refer to the drawings for clarity. are described herein for and are not intended to limit the actual device or system or the use of the device or system. Many of the devices, products or systems described herein can be used in a number of directions and orientations. Descriptors of orientation, as used herein with reference to cell culture devices, often refer to the orientation when the device is oriented for the purpose of culturing cells within the device.

Unless expressly stated otherwise, any method described herein is not intended to be construed as requiring that the steps be performed in a particular order. Thus, if a method claim does not actually list the order in which the steps must be followed, or unless it is specifically stated otherwise in the claims or description that the steps are to be limited to a specific order, then any specific order is inferred. not intended Any single or multiple feature or aspect recited in any one claim may be combined with or substituted for any other feature or aspect recited in any other claim or claims.

It is also noted that enumeration herein means a component that has been “configured” or “adapted” to function in a particular way. In this regard, such components are "configured" or "adapted" to implement a particular feature or function in a particular way, where such recitation is a structural recitation as opposed to an intended use recitation. More specifically, references herein to the manner in which a component is “constructed” or “modified” refer to the existing physical condition of the component and, as such, should be regarded as a clear recitation of structural features of the component.

Where various features, elements, or steps of a particular embodiment can be disclosed using the transitional phrase “comprising”, optional embodiments include those that may be described using the transitional phrases “consisting of” or “consisting essentially of.” It will be understood that it is implied. Thus, for example, an implied alternative embodiment for a cell culture device comprising a structure defining a plurality of wells is an embodiment where the cell culture device consists of a structure defining a plurality of wells and a cell culture device It includes embodiments where it consists essentially of a structure defining a plurality of wells.

In various embodiments, the present disclosure describes a device, such as a cell culture device, comprising a substrate defining wells (eg, microwells). A well includes a sidewall (or sidewalls), a well-bottom (or nadir) and an open top (eg, top opening). In an embodiment, the well is configured to contain an aqueous liquid composition, eg, a composition used for cell culture or cell assay. For example, an aqueous liquid composition may include cell culture media, buffers or other solutions or mixtures used in cell assays.

Embodiments described herein find use with any device that includes, for example, a small (eg, microscale) well or other vessel or chamber configured to contain a liquid. In certain embodiments, wells of a device described herein are used for cell culture. More specifically, the device and microwells therein are used for 3D cell culture of cell aggregates or spheroids.

There are several different geometries that have been used for culturing cells as aggregates. In some embodiments, cell aggregates are clusters of cells, embryoid bodies, or spheroids. A common geometry for forming cell aggregates is the hemisphere found in round bottom-well microplates. In some embodiments, non-adherent surfaces are used to prevent cells from adhering to the surface. The non-adherent material may be applied after the well or chamber (eg, microplate) is fabricated, or the well or chamber material may have inherent non-adherent properties.

1 is a schematic diagram of a representative embodiment of an array of wells 100 , showing individual wells 115 . In the embodiment illustrated in FIG. 1 , well 115 has a mouth 110 . Mouth 110 is the region of the upper part of the well, adjacent to the upper opening 111 of the well 115, before the well shrinks to form the well-bottom to which cells settle to form a spheroid, Provide more open areas. In embodiments, the mouse 110 may be conical (wider at the top of the mouse than at the bottom of the mouse) and annular (with rounded wells, as shown in FIGS. 1A and 2A ). In additional embodiments, if the well has a round opening but is parabolic in shape, for example, as shown in FIG. 3A , the mouse 110 may be parabolic in shape or as shown in FIGS. 27 and 28 . , a mouse can extend into each well 115. In an embodiment, there is no mouse. The presence of mouse structures may serve two functions. First, the mouse widens the well's opening, and allows the liquid introduced into the well's opening to flow down the well. This promotes the aggregation of cells at the bottom of the well and promotes the formation of spheroids in culture. Additionally, the mouse creates a transition between the annular inner surface of the mouse and the inner surface of the well, thereby providing a geometrical feature that can prevent entrapment of air in the well. The presence of a 90 degree angle between the top of the well and the sidewall of the well may provide a location for the formation of air bubbles. Mice provide a transition between the top of the well and the sidewall that is not at a 90 degree angle, thereby reducing the formation of air bubbles in wells with a mouse structure.

Dimensions for use in agglutination cell culture techniques may be on the order of micrometers to millimeters (eg, 100 μm to 50 mm). Well-containing devices for cell culture are sold by many different manufacturers (eg, Corning, Nunc, Greiner, etc.). A "microwell" is generally of the order of magnitude in micrometers (eg, ≤1 mm, ≤500 µm, ≤400 µm, ≤200 µm) or several millimeters (eg, ≤10 mm, ≤5 mm, ≤3 mm, etc.) , and also used to grow cells into aggregates. In some embodiments, microwells provide a confinement for 3D cell culture. In any suitable embodiment herein, the term “well” refers to, except where otherwise stated or indicated by context (e.g., a well is described as having a well-bottom comprising a plurality of microwells). if available) covers the use of microwells. Wells intended to fall outside the microwell dimensions may be referred to as "macrowells" or simply "wells". In some embodiments, the well height or well-depth (e.g., from the top opening to the well-bottom) equals 100% or more of the well-diameter at the top opening (e.g., 100%, 110%, 120 %, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, or any range in between). In some embodiments, the well-depth (e.g., from the upper aperture to the well-bottom) equals at least 100% of the well-diameter at the midpoint between the upper aperture and the well-bottom (e.g., 100% , 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375 %, 400%, or any range therebetween).

One of the most commonly used microwell products is the commercially available "Aggrewell" plate (sold by Stem Cell technologies), which has the shape of an inverted pyramid with a diameter of 400 or 800 micrometers arranged at the bottom of a standard format microplate well. provides the geometry of Another geometry for growing cells as aggregates is the "Elplasia" microplate with "microspace cell culture" (Kuraray); These plates have square microwells of 200 micrometers in diameter arranged at the bottom of standard format microplate wells to allow cells to aggregate. The various parameters, dimensions, and methods of making microwells for culturing cells in aggregates are understood in the art (US 2004/0125266; US 2012/0064627; US 2014/0227784). ; WO2008/106771; WO2014/165273; the entire contents of which are incorporated herein by reference). US Patent No. 6,348,999 describes micro relief elements and how they are constructed, without stating the purpose of these structures other than a polymer lens array. U.S. Patent Nos. 5,151,366, 5,272,084, and 6,306,646 describe containers having various types of micro-relief patterns to increase the surface area for cell attachment on a substrate, and methods for producing culture patterns, but the patterns themselves will not be conducive to the formation of cell aggregates. Other devices, compositions, reagents and methods have been described in the art, see, for example, US 2014/0322806; U.S. Patent No. 8,906,685; Haycock. Methods Mol Biol. 2011; 695:1-15; US Published Patent No. 2014/0221225; WO 2014/165273; U.S. Patent Publication No. 2009/0018033; The entire contents of these are incorporated herein by reference.

Some commercially available well geometries are conducive to the formation of cell aggregates, but not necessarily conducive to "confinement". If aggregated cells are not restrained, they will usually grow as large as their surroundings will allow. Aggregates of cells greater than 150 to 400 micrometers in diameter (depending on the cell type) may form necrotic cores. For example, necrosis occurs because cell clumps are so large that diffusion of nutrients into the center of the aggregate and diffusion of metabolic waste products out of the center of the aggregate is restricted. In some embodiments, to create confinement, the microwell geometry is very similar in diameter to the size of the maximum desired cell aggregate (e.g., 50%, 40%, 30%, 20%, 15%, 10% , 5%, 2%, 1%, or an appropriate range therein), and a depth of at least 1.5 to 2 times the diameter (e.g., 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 , 2.5, 2.6, 2.6, 2.8, 3.0, 3.5, 4.0 or an appropriate range therein). In some embodiments, the confinement well geometry also allows exchange of liquid culture medium via perfusion or manual pipetting without lifting the cell spheroids outside the confinement well.

Existing devices (eg, microwell format microplates or other containers with microwells) exhibit design flaws that negatively impact the use of these devices for the formation of 3D cell aggregate structures. Although handling of microwell format microplates is fairly straightforward, it is often problematic if air is not displaced from the microwells upon introduction of a liquid (eg, medium). Air entrapment is a common problem in microplate wells with geometries as large as the 384-well format, especially if the wells are circular. The surface tension of aqueous liquids is so strong that they hold the droplets in spheres. A spherical drop can block a circular hole of similar size (eg, a geometry that is a well cross-section). The presence of air in the well will negatively affect and/or inhibit cell culture within the well.

Growing spheroids in a cell culture vessel at high density on a substrate having an array of spheroid formation wells with non-adherent surfaces requires a culture surface that balances many variables. For example, balancing the maximum achievable spheroid density, the ability to retain the spheroids in position during fluid exchange activities and remove them if desired, and avoid entrapment of air in the spheroid forming wells when the vessel is filled. An achievable design is highly desirable to avoid the difficulties of working with such vessels. Embodiments herein address the air entrapment problem of traditional wells by providing a well geometry that will help displace air within the microwells and allow entry of liquid into the microwells, while maintaining confining dimensions. For example, a square upper well with a round well-bottom geometry is sufficiently less likely to entrap air when liquid is added because air can rise over the corner of the well around an aqueous droplet. In some embodiments, the inclusion of various structures (eg, columns, corrugations, corners, ridges, crevices, etc.) extending from the opening of the well to the bottom of the well provides a path for air to escape upon introduction of liquid into the well. to provide. In some embodiments, in addition to the features described herein, also include geometries, materials, etc. described in and/or understood in the art.

To avoid the problem of air entrapment in high-density spheroid growth substrates, one design feature commonly utilized is the avoidance of sharp corners or step changes in the substrate geometry, particularly orthogonal to the flow path of the liquid across the surface. For example, an Aggrewell plate has walls that fall at an angle of nearly 90 degrees. This promotes the escape of the liquid from the surface as the container is filled, leaving wells filled with air.

Provided herein are surface geometries that maintain well features (eg, rounded well-bottoms) that promote growth and maintenance of high-density discrete spheroids while addressing the problem of air entrapment during liquid introduction.

In some embodiments, well-shape inversion is utilized to alleviate problems with air-venting upon introduction of liquid into the well. For example, in some embodiments, a circular cross-section well-bottom (or lower portion of a well) is utilized for spheroid formation. However, circular cross-sections can be particularly problematic for air evacuation without pocket formation. To alleviate this problem, the well is formed with a circular bottom well cross-section and a non-circular (eg, triangular, square, rectangular, pentagonal, hexagonal, etc.) top opening. In this embodiment, the sidewall transitions from a non-circular (eg, polygonal) top opening to a circular well-bottom. In some embodiments, the conversion is performed so as not to introduce any interfering, jagged, or horizontally-existing sidewall features that could result in a bubble "hanging up" that "falls" the well upon introduction of liquid into the well. , it is done gradually. In some embodiments, the corners of the side walls created by the transitioning walls and the non-circular (eg, polygonal) shape of the top opening provide pathways for liquid entry and/or air exit.

In some embodiments, the well geometry includes capillary structures (e.g., including mouths, ridges, clefts, rounded or parabolic top openings, etc.) in the well walls to facilitate the evacuation of air upon introduction of liquid into the wells. do. FIG. 1B is a plan view, taken at line B-B of FIG. 1A , illustrating ridge 170 . As shown in FIG. 1B , a ridge is a ridge or projection from the mouth 110 or side wall 113 of a well. In an embodiment, the ridge extends the length of a microwell from the top opening 111 to the bottom 116 of the well. In additional embodiments, the ridge extends from the top of the mouth 111 to the bottom of the mouth 112 . The sharp angles formed on either side of the ridge 170 create capillary forces on the aqueous fluid to provide fluid entry into the microwells without entrapment of air. FIG. 2B is a top view of the array of wells 100 shown in the cross-sectional view of FIG. 2A. 2B illustrates cleft 270 . As shown in FIG. 2B, the crevice is a depression in the sidewall 113 of the well 115. The sharp angles formed on either side of the crevice 270 create capillary forces to allow aqueous fluid to flow into the microwells.

3A and B are schematic diagrams of another representative embodiment of an array 100 of wells. 3A is an example of a cross section. FIG. 3B is a top view of a representative embodiment of an array of wells, taken at line B-B in FIG. 3A. 3A and 3B show that each well 115 can have one or more ridges 170 or crevices 270, and the ridges 170 or crevices 270 can be arranged in an array within the well 115. can As shown in FIGS. 3A and 3B , in an embodiment, the radial distribution of ridges and/or fissures is envisaged. The number of capillary structures is not limited to one per microwell. In some embodiments, a larger number of capillaries increases the rate of fluid entry into the microwells.

Figures 3a-d show a number of cells (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 24, 28, 32 or any range therein) in a single well. ) shows the inclusion of vertically-oriented capillary structures. Features can be regularly-spaced (shown in Figure 3), irregularly spaced, grouped/bundled, etc. In some embodiments, the capillary structure extends from the top opening of the well to the well-bottom. When multiple capillary structures exist within a single well, the multiple features may be of different types (eg, ridgelines and/or clefts) and may include other shapes (eg, square, round, etc.) .

3C illustrates that the array of wells 100 can have a sinusoidal or parabolic shape. This shape, in embodiments, creates a rounded top edge or well edge that reduces entrapment of air at a sharp corner or 90 degree angle 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, well 115 has a top of the well with a top diameter (D _top ), a height from the bottom of well 116 to the top of the well (H), and the top of the well and the bottom of the well. (116) has a diameter of the well (D _{half - way} ) at an intermediate height between

3D is a schematic diagram of an array of wells, as described in FIGS. 1-3 and embodiments. 3D illustrates a plurality of microwells 115 arranged in an array on a substrate 1114. 3D also shows a plurality of spheroids 500 present in a plurality of microwells 115 .

4A and 4B are schematic diagrams showing another representative embodiment of an array of spheroid-containing cell culture compartments, or wells. 4A is a plan view of a corrugated embodiment of an array of wells, and FIG. 4B is a partial cut-away view of the same embodiment. In the embodiment shown in FIGS. 4A and 4B , the wells are not separated but allow the flow of liquid between the wells. As shown in FIGS. 4A-B , representative embodiments are illustrated in which corrugated sidewalls 403 are arranged to create microwells 401 in gaps between corrugated sidewalls 403 . The corrugated sidewalls shown in FIGS. 4A and 4B are surrounded by a frame 402 and are spaced apart from each other and in close contact with each other (e.g., contact or non-contact) within the heat generating cycle of the microwells 401. have an area In some embodiments, a microwell depression is placed at the base of each segment defined by the more distant wall (eg, to accommodate the spheroid 500). 4C and 4D are additional schematics of corrugated microwell array embodiments. 4C is a plan view, and FIG. 4D is a perspective view of an embodiment. In an embodiment, as shown in Figure 4d, there are no frames. 4C and 4D illustrate a corrugated or wavy sidewall 403 defining a space with a geometry 401 suitable for inducing spheroid formation. 4c and 4d also show a spheroid 500 present in the well. When liquid is introduced into the embodiment shown in FIGS. 4A and 4B , the displaced air can migrate out of the localized area through areas where the walls are in close proximity to each other. Movement of fluid and air to and from the wider well area is facilitated by the narrow section to avoid air entrapment. These wrinkles also provide a constricted growth zone to promote the formation of spheroids. That is, wrinkles are both capillary structures and spheroid-derived geometries.

5A and 5B show a schematic plan 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 an embodiment, the top 510 of the post 501 may be flat (as shown in FIG. 5A ). In this embodiment, the convex walls created by the columns 501 create very sharp angles or discontinuities at the junctions between the column-like sidewalls. 5B shows a microwell depression surrounded by pillars to create a confinement geometry suitable for inducing the formation of spheroids 500 in culture. Air exits through the space 505 between the pillars 501 and fluid enters. The column 501 has a top 510 . In an embodiment, the column top 510 can be rounded, as shown in FIGS. 5B, 5D and 5E, which will result in a well having a sinusoidal or parabolic shape as shown in FIG. 3A. Pillars also provide a constricted growth zone to promote the formation of spheroids. That is, the pillars are both capillary structures and spheroid-guided geometries.

For example, ridges, crevices, ridges, divots, top openings or open ring structures of mouth structures, columns, discontinuous walls, mouth structures, parabolic or sinusoidal well shapes, rounded well openings, or smooth sidewalls of wells. These structures, including obstructions at the inner surface, or any combination of these features, are capillary structures. The capillary structure also provides an escape route for any air that may be trapped after adding liquid. In some embodiments, discontinuous walls, walls containing ridges or crevices or other features that impede the smoothness of the sidewalls of the wells, are used to avoid entrapment of air by providing ventilation locations within the wells during filling.

In some embodiments, the capillary structure extends along the vertical length of the wall of the well. In some embodiments, the capillary structure extends to or over the top opening of the well. In other embodiments, the capillary structure is located near the top opening of the well (e.g., <0.1, 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 10 μm from the top opening). or 20 μm (or ranges therebetween)). In some embodiments, the capillary structure extends to the bottom of the well. In other embodiments, the capillary structure is located near the well-bottom (e.g., <0.1, 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 10 μm from the well-bottom). or 20 μm (or ranges therebetween)).

In some embodiments, the capillary feature provides the advantage of providing a path for liquid to enter the well to move without trapping gas within the well. In some embodiments, the capillary structure provides a pathway for air to exit the well relative to the introduction of liquid entering the well. The above technology is not limited to any particular mechanism of action for preventing air entrapment, and an understanding of the mechanism is not essential to practicing the present invention.

Despite well geometries configured to prevent such entrapment, when rapid vessel filling or other fluid-introduction induces air entrapment, the capillary feature allows the movement of liquid beneath the entrapped air, thereby releasing air from the well. For example, in the corrugated embodiment shown in FIG. 4, liquid and air can flow from one well region through an open well structure to the next well region through a narrow section of the array. Alternatively, in the pillar embodiment shown in FIG. 5, liquid or entrained air may exit the wells through the spaces between the pillars. In some embodiments, the downward force of the liquid through the capillary structure serves to separate the air from the well walls, enveloping the air in liquid so that air pockets rise from the well as bubbles.

A variety of other homeotropic-oriented structures are used with capillary features. For example, in certain embodiments, the features are raised ridges (eg, as shown in FIGS. 1A and 1B ) or recessed grooves or fissures (eg, as shown in FIGS. 2A and 2B ). am. The ridgelines and crevices used in the embodiments herein are not limited to the physical geometries shown in the figures. In some embodiments, the feature extends vertically along the sidewall of the well, rather than transverse in a horizontal direction. In most situations, the horizontal structural feature promotes air entrapment within the well in a manner similar to steep angles used in existing well geometries. Suitable cross-sectional geometries for ridges are shown in FIG. 6 and include: round (FIG. 6A), angular (FIG. 6B), acicular (FIG. 6C), half-hexagonal (FIGS. 6D and 6E), and the like. Suitable cross-sectional geometries for clefts are shown in FIG. 7 and include: round ( FIG. 7A ), angular ( FIG. 7B ), acicular ( FIG. 7C ), half-hexagonal ( FIGS. 7D and 7E ), and the like. Ridges and/or crevices may have any suitable cross-sectional dimension (eg, 0.1-20 micrometers (eg, 0.1, 0.2, 0.5, 1, 2, 5, 10, 15, 20, or any in between) It may be a dimension) having a width, length, etc. of a suitable range of). Engineering and microfluidic principles are ridged and/or crevice shapes and/or crevice shapes to facilitate the introduction of liquid and the evacuation of air without the formation of air pockets below the liquid surface and/or to facilitate the removal of trapped air pockets. It can be used in combination with the embodiments herein to optimize dimensions.

In some embodiments, the movement of liquid into and out of the well is facilitated by the discontinuous sidewall geometry. Discontinuous well geometries take the form of discontinuities in the sidewalls of the wells. Examples of discontinuous sidewall geometries are shown in the "wave wall" or "corrugated" geometry of FIG. 4 and the "pin wall" or "column wall" These geometries are representative only; Other sidewall orientations that introduce gaps or other discontinuities in a portion (eg, top) of the sidewall(s) may be used in the embodiments herein. In these geometries, the obstruction(s) in the walls cause low viscosity air to move rapidly out of the well as the fluid enters the vessel. In some embodiments, the discrete geometry preserves avoidance of sharp angular changes in substrate wall features (eg, avoidance of features that create horizontal obstacles). In some embodiments, discontinuous sidewall geometries are used in conjunction with well-shaped transitions and/or capillary wall structures to aid bubble release and/or air release upon liquid introduction.

In some embodiments, one or more wells have a concave surface, such as a semi-spherical surface with a rounded bottom or a conical surface, and similar surface geometries, or combinations thereof. The wells and well bottoms may ultimately be end, end, or finished with rounded or curved surfaces, concave frusto-conical relief surfaces, or combinations thereof, such as, for example, dimples or wells, and the like. there is. Other types and configurations of spheroid-serving wells are described in commonly-assigned U.S. Patent Application Serial No. 14/087,906, which is incorporated herein by reference in its entirety without conflicting with the present disclosure. get mixed up In an embodiment, the well-bottom is flat or reaches a point. The bottom-well may have other suitable shapes or dimensions. For example, in embodiments, the well bottom has a rounded or curved surface, or the well bottom has structures such as dimples, pits and the like, concave frustoconical relief surfaces, dimples, or pen-tip regions, or combinations thereof. , which promotes the formation of spheroids by providing a constricted growth zone. That is, a rounded or curved or dimpled well bottom or pen tip area or crease or column is a spheroid derived geometry.

Representative well geometries and sizes are shown, for example, in FIGS. 1 and 2 . In some embodiments, a well 100 described herein is about 100 micrometers to about 2000 micrometers, for example 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800 or 2000 micrometers, and any two ranges between the foregoing values (e.g., 200-1000 μm, 200-750 μm, 300-750 μm, 400-600 μm, etc. ), and has a well-bottom diameter (130/230). This diameter dimension regulates the size of the spheroid grown inside the spheroid to keep the cells healthy inside the spheroid. That is, these dimensions provide a constricted growth zone to promote the formation of spheroids. That is, these dimensions are the spheroid derived geometry.

In some embodiments, wells (100/200) described herein range from about 100 micrometers to about 2000 micrometers, for example, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700 , 800, 900, 1000, 1200, 1400, 1600, 1800, or 2000 micrometers, and an upper opening cross-sectional dimension (120/220) including any two ranges between the foregoing (e.g., diameter or width). have In some embodiments, the well 100 has a range of about 500 micrometers to about 1500 micrometers from the top opening to the well-bottom, for example 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 micrometers. , and a depth (160/260), including any two ranges between the aforementioned values. In some embodiments, a well (100/200) is in a range from about 50 micrometers to about 500 micrometers, for example 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 micrometers, and any of the above values It has an upper portion with a depth (140/240) encompassing the range between any two. In some embodiments, the well 100 is between about 100 micrometers and about 1400 micrometers, for example, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 , 1200 or 1400 micrometers, and a lower portion with a depth (150/250), including any two ranges in between the aforementioned values. Of course, other suitable dimensions may also be used.

In some embodiments, devices and wells (e.g., microwells) may include additional features to provide special functionality, for example related to 3D culture of cells within the microwells. The following paragraphs address these features that may be used in combination with the foregoing embodiments.

In some embodiments, all or part of the well's sidewall and/or well-bottom are gas permeable. In some embodiments, the gas permeability allows the movement of oxygen and other gases to the well to dissolve into the liquid or medium contained within the well. The permeable sidewall, well-bottom, or part thereof does not allow the formation of air pockets or bubbles within the well liquid.

In some embodiments, a cell culture device having a structured surface defining a plurality of gas permeable wells is provided. In some embodiments, the well may include an exterior surface defining an exterior surface of the device. In some embodiments, a well may include an external or non-cultivating surface in communication with the exterior of the device. In some embodiments, provided herein is a cell culture device having, among other things, a plurality of stacked cell culture compartments, each having a structured surface defining a plurality of gas permeable wells. In some embodiments, in various embodiments the well is in gas communication with the exterior of the device, eg, indirectly through a vent or through a conduit space, or directly through an exterior wall.

In some embodiments, the well is configured such that cells cultured in the well form spheroids. For example, in some embodiments, the well is non-adherent to the cells such that the cells in the well bind to each other (eg, to form a spheroid). The spheroid expands to the size limits imposed by the geometry of the well. In some embodiments, the wells are coated with an ultra-low binding material to make the wells non-adherent to the cells.

In contrast to two-dimensional cell culture, in which cells form a monolayer on the surface, formation of three-dimensional (3D) cell aggregates, such as spheroids, increases the density of cells grown in a cell culture device, which in turn increases the density of the cells grown in the device. increase the nutrient demand of cultured cells in Because metabolic gas exchange can occur through gas permeable wells in which cells are cultured, the media volume in a cell culture device can be larger than in a device where metabolic gas exchange is essentially limited to diffusion through the cell culture medium. Thus, larger heights, and thus volumes, of cell culture medium can be used with the cell culture devices described herein.

In some embodiments, cells are cultured in wells of a device described herein where the cell culture medium is at least 2 mm above the cells. In some embodiments, the cell culture medium is at least 5 mm taller than the cells. A maximum cell culture medium height of 2 mm to 5 mm is generally used when metabolic gas exchange is necessarily restricted through the medium, for example, the substrate or surface on which the cells are cultured, or a surface near it, is inhospitable to metabolic gases. If it is permeable or relatively impermeable (eg, compared to the cell culture medium), it is considered an upper limit for medium height.

For the purpose of effective metabolic gas exchange, in some embodiments, the cell culture medium in the device is at any height above the cells, such as 2 mm above the cells, or 5 mm above the cells or 10 mm above the cells. is maintained in culture in the wells of the device described herein. However, one skilled in the art will understand that as the height of the cell culture medium within the device increases above the cells, the hydrostatic pressure exerted on the cells increases. Thus, there may be practical limitations 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 a well of a product described herein is between 5 mm and 15 mm, 6 mm and 15 mm, such as 5, 6, 7, 8, 9, 10, 15 or 20 mm, 5 mm. to 10 mm or 6 mm to 10 mm, including ranges from 5 mm to 20 mm, and any two ranges between the aforementioned values.

In certain embodiments, cell culture substrates or layers having non-cultivation surfaces in gas communication with the outside of the device may be adapted to have structured surfaces defining gas permeable wells as described herein. Examples of such cell culture devices include T-flasks, TRIPLE-FLASK cell culture vessels (Nunc., Intl.), HYPERFLASK cell culture vessels (Corning, Inc.), CELLSTACK culture chambers (Corning, Inc.), CELLCUBE modules ( Corning, Inc.), CELL FACTORY culture device (Nunc, Intl.), and WO 2007/015770, US Patent Publication No. 2014/0315296, US Patent No. 8,846,399, US Patent No. 8,178,345, and US Patent No. 7,745,209 The patents and published patent applications are incorporated herein by reference in their entirety to the extent that they do not conflict with the disclosure herein.

In some embodiments, gas-permeable/liquid-impermeable materials are used herein in the manufacture of cell culture devices or parts thereof (eg, wells, microstructures, etc.). polystyrene, polycarbonate, ethylene vinyl acetate, polysulfone, polymethyl pentene (PMP), polytetrafluoroethylene (PTFE) or compatible fluoropolymer, silicone rubber or copolymer, poly(styrene-butadiene-styrene), or polyethylene or Any suitable gas-permeable/liquid-impermeable material may be used, such as a polyolefin, such as polypropylene, or a combination of these materials. The substrate may be formed of any suitable material having suitable gas permeability to at least a portion of the well. 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 a thickness of up to 40 mm. PMP can achieve sufficient gas permeability at up to about 0.1 mm thick. In some embodiments, the PMP has a thickness ranging from about 0.02 to 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, open frames, standoffs, or the like may be used to support the substrate underneath. In embodiments, the well thickness may be 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20 or 40 mm, and a thickness including any two ranges in between the aforementioned values. In an embodiment, the well has an oxygen transmission rate through the gas permeable polymeric material of at least 2000 cc/m 2 /day. In some embodiments, the well has a gas permeability through the substrate greater than or equal to 3000 cc/m 2 /day. In some embodiments, the well has a gas permeability through the substrate of greater than 5000 cc/m 2 /day.

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

In some embodiments, the thickness of the well substrate material is adjusted to allow for optimized gas exchange. In some embodiments, the well-thickness is between 10 and 100 μm (e.g., 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm , 80 μm, 90 μm, 100 μm and any range therebetween). Experiments performed during development of the embodiments herein show higher numbers of viable cells generated with thinner well-thickness (eg, 17 μm, > 30 μm, > 57 μm).

8 is an illustration of an embodiment of including a substrate having an array of microwells incorporated as a surface of a cell culture device. In FIG. 8 , the cell culture device is a flask 800 . However, in an embodiment, the substrate is a cell including but not limited to a multiwell plate, flask, dish, tube, multi-layer cell culture flask, bioreactor, or any other laboratory vessel intended for growing cells or spheroids. It should be understood that it may form part of any kind of culturing device. A substrate having an array of microwells may be a gas permeable material. FIG. 9 shows an enlarged view of a substrate, micropatterned with an array of microwells, forming the lower surface of the flask shown in FIG. 8 .

Referring to the embodiments illustrated in FIGS. 8 and 9 , the illustrated device is a flask or housing containing a cell culture chamber 850 . The housing includes a substrate having an array of microwells (shown in FIG. 9 ). The housing also has an upper surface 815 , one or more sidewalls 820 extending from the structured surface 110 to the upper surface 815 . In some embodiments, housing 850 includes a single enclosure sidewall, such as a cylindrical wall or the like. Additional embodiments of such devices are disclosed, for example, in commonly-assigned U.S. Provisional Patent Application Serial No. 62/072,015, which is incorporated herein by reference in its entirety to the extent that it does not conflict with this disclosure. get mixed up

The housing includes port 860. 8 shows an opening with a screw top 861. However, in embodiments, the housing may have any type of port that allows liquids and cells to enter and exit the housing. Port 860 can be on the sidewall, top surface 815 or cell culture surface 110 . Port 860 can be connected to tubing or other connections to introduce or remove cells and cell culture medium into or out of cell culture chamber 850 .

In an embodiment, although the housing shown in FIG. 8 illustrates a fixed sidewall 820 , the sidewall may be flexible or expandable to allow variable volume of cell culture medium into the cell culture chamber 850 and can be folded When additional cell culture medium is introduced into the cell culture chamber 850, through port 860, the flexible sidewall 820 can be extended and cultured from the cell culture chamber 850, through port 860. When the medium is removed, the flexible sidewall 820 may collapse. In some embodiments, sidewall 820 and top 815 are formed of a bag. Additionally, the cell culture chamber 850 can be filled with any volume of cell culture medium up to a fixed volume housing. In some embodiments (not shown), all or nearly the entire internal volume is filled with cell culture medium.

In the embodiment shown in FIG. 8 , the volume of cell culture medium in the cell culture chamber 850 is at the height of the medium above the cells (H _m ). As noted above, the height of the medium (H _m ) in a housing as described herein above the cultured cells may be higher than would be possible if the wells were not gas permeable. In some embodiments, cell culture chamber 850 is filled to its capacity for the purpose of culturing cells within the device.

FIG. 10A shows HT29 cell spheroids 500 inside microwells 110 of a micropatterned T25 spheroid forming flask, as shown in FIG. 9 . 10B shows spheroids 500 harvested from a micropatterned T25 spheroid forming flask, according to Example 2 described below. 11A and B are photomicrographs illustrating a wide range of size distributions of 3D aggregates (human ESC cells in FIG. 11A and mouse ESC cells in FIG. 11B) formed on NUNCLON SPHERA™ low binding surfaces, available from Nunc. The NUNCLON SPHERA™ low binding surface has a low cell binding surface finish, but lacks the geometry disclosed herein that allows for the formation of uniform spheroids.

12 is an illustration of a method of fabricating a multiwell array substrate, according to an embodiment, and as described in Example 1 below. 12 shows a high temperature embossing/thermoforming process, other methods of making microwell arrays according to embodiments are contemplated, including coining, injection molding, embossing, and other methods known in the art. .

13, 14a and b, and 15 are graphs comparing viable cell counts ( FIGS. 13 and 14a ) and cell productivity ( FIGS. 14b and 15 ) for substrates having arrays of microwells compared to flat surfaces. The gas permeability of the well may depend in part on the thickness of the substrate depending on the material of the substrate and the well. In an embodiment, the thickness of the sidewalls and bottom of the wells in a substrate having a microarray of wells can be constant and relatively thin. Alternatively, in an embodiment, the walls of a well in an array of microwells may be relatively thick proximate the opening to the well and relatively thinner at the bottom of the well. Or, in an embodiment, the walls of the wells in the array of microwells can be relatively thinner proximate the opening into the well and relatively thicker at the bottom of the well. Depending on the material used and the thickness used, the substrate having the array of microwells may be gas permeable for purposes of this disclosure.

15 is a graph showing the total protein titer released from MH677 cells cultured on substrates with microwell arrays compared to flat surfaces. This data is discussed in Example 2 below.

Although the device shown in FIG. 8 can be a hard-sided flask or a soft-sided cell culture flask, it includes a structured microwell array having a surface that defines an exterior surface of the cell culture device or is in gas communication with the exterior of the device. It will be appreciated that any other cell culture device that does this may have a substrate with an array of microwells formed of a gas permeable material as described herein.

A structured surface of a cell culture device having an array of microwells as described herein may define any suitable number of wells, which may be of any suitable size or shape. Wells define a volume based on their size and shape. In many embodiments, one or more or all wells can rotate symmetrically and/or symmetrically about a longitudinal axis. In some embodiments, the longitudinal axes of one or more or all wells are parallel to each other. The wells may be spaced uniformly or non-uniformly. 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 relative to the well is configured to correct refraction of light passing into the inner surface and out of the outer surface. In some embodiments, correction is achieved by adjusting the thickness of the substrate material forming the well. In some embodiments, the thickness of the substrate material proximate the well-bottom (or nadir) is greater than the thickness of the substrate material proximate the sidewalls and/or top openings. In some embodiments, the thickness of the substrate material progressively decreases from a maximum value at the nadir of the well bottom to a minimum value at the top aperture. For example, shapes and thicknesses are disclosed in commonly-assigned U.S. Provisional Patent Application Serial No. 62/072,019, the entire contents of which are incorporated herein by reference to the extent that they do not conflict with the present disclosure. do.

For example, the combination of non-adherent wells, spheroid-guided well geometry, and gravity can define a confinement volume within which growth of cultured cells in the well is limited, which is defined by the confinement volume. It results in the formation of a spheroid with dimensions.

In some embodiments, the inner surface of well 2115 is non-adherent to cells. The wells may be formed of a non-adherent material or may be coated with a non-adherent material to form a non-adherent well. Examples of non-adhesive materials include perfluorinated polymers, olefins or similar polymers or mixtures thereof. Other examples include agarose, non-ionic hydrogels such as polyacrylamide, polyethers such as polyethylene oxide and polyols such as polyvinyl alcohol, or similar materials or mixtures thereof. For example, a combination of non-adherent wells, well geometry, and gravity can induce cells cultured within wells to self-assemble into spheroids. Some spheroids are able to retain differentiated cell functions that represent a response rather close to in vivo compared to cells grown in monolayers.

In some embodiments, one or more wells have a concave surface, such as a hemi-spherical surface, a conical surface with a rounded bottom, and similar surface geometries or combinations thereof. Wells and well bottoms may ultimately be end, end, or finished with rounded or curved surfaces conducive to spheroids, concave frusto-conical relief surfaces, or combinations thereof, such as dimples, pits, and the like. Other types and configurations of gas-permeable spheroid-conducive wells are described in commonly-assigned U.S. patent application Ser. No. 14/087,906, which is incorporated herein by reference in its entirety to the extent that it does not conflict with the present disclosure. get mixed up

In some embodiments, the well-bottom is flat or reaches a point. The bottom-well may have other suitable shapes or dimensions.

In some embodiments, wells 115 described herein are in the range of about 200 micrometers to about 500 micrometers, for example, 200, 250, 300, 350, 400, 450, or 500 micrometers, and any value between the foregoing. It has a diameter dimension (w) in two ranges. These diameter dimensions can adjust the size of the spheroids grown therein so that the cells inside the spheroids remain healthy. In some embodiments, well 115 is between about 100 micrometers and about 500 micrometers, for example, between 100, 150, 200, 250, 300, 350, 400, 450, or 500 micrometers, and any two values in between. It has a height (H) in a range that includes a range of . Of course, other suitable dimensions may also be used.

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

18 is a side view illustrating a specific example of a cell culture device including a porous membrane support. Various embodiments of a cell culture device 2100 incorporating a porous membrane support 2500 are shown in FIGS. 18-20. A porous membrane support 2500 is disposed across the housing to the device (eg, coupled to one or more sidewalls 2120) to partition the interior of the housing into separate incubation chambers 2152, 2154. The first incubation chamber 2152 includes a substrate defining a structured surface defining a gas permeable well 2200 in gas communication with the exterior of the device. The top of chamber 2152 is bounded by porous membrane 2500. The lower portion of the second incubation chamber 2154 is bounded by a porous membrane 2500. Thus, a first population of cells 2200 can be cultured in a first chamber 2152 in a well 2115 of a structured surface formed by a substrate 2110, and a second population of cells 2202 can be a porous can be cultured in the second chamber 2154 on the membrane 2500.

The device shown in FIGS. 18-20 includes a first port 2162 in communication with a first chamber 2152 and a second port 2164 in communication with a second chamber 2154 . In additional embodiments, either the first chamber 2152 or the second chamber 2154 can optionally have additional ports (outlet ports, not shown) to allow flow of liquid through the chambers. Ports 2162 and 2164 can be, for example, ports similar to those discussed and illustrated with respect to FIGS. 21-23 below (eg, port 2160). Ports 2162 and 2164 can be on the same side of device 2100, or can be on opposite sides, or provide separate access to the chambers or chambers 2152 and 2154, as shown. ) can be oriented in any other suitable way to flow through.

In FIG. 18 , the device is shown operating without a headspace (volume filled with cell culture medium). In Figures 19-20, the device is shown operating with a head space (not filled to volume with culture medium). Due to the porous nature of the support 2500, the chamber 2152 remains filled with culture medium while it can be operated with or without a headspace. Due to the gas permeability of the well 2115, the height of the medium above the cells 2200, for example, as described above, may not be of critical concern. However, if the housing is 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 Figures 18-20, co-culture of more than one cell population is contemplated. For example, a first cell population can reside in the first chamber 2152 while a second cell population can reside in the second chamber 2154. These cell populations can be separated by permeable membrane 2500. This allows chemical communication between the first population of cells and the second population of cells. In an embodiment, one or both of these cell populations may be spheroids. For example, as shown in FIG. 20 , a first population of spheroid cells 2200 can grow in the first chamber 2152 to form spheroids due to the spheroid-guided geometry of the cell culture substrate. while the second population 2202 of spheroid cells will also grow in the second chamber 2154 to form spheroids due to the spheroid-guided geometry of the cell culture substrate present in the second cell culture chamber. can Via porous membrane 2500 , the second population of spheroid cells 2202 is in chemical communication with the first population of spheroid cells 2200 . In this way, it is possible to co-culture two separate populations of spheroid cells while they are chemically linked to each other. Alternatively, in an embodiment, as shown in FIG. 18 , the first cell population can be spheroid cells growing in the first cell culture chamber 2152 (and the second cell culture chamber does not have a spheroid inducing geometry). ), a second cell population that is not spheroid cells can grow in the second cell culture chamber 2154, while the presence of the porous membrane 2500 allows the first and second populations of cells to be in chemical communication with each other. let it As shown in FIG. 19 , the second population of cells can grow in the presence of a head space, or as shown in FIG. 18 , the second population of cells can grow without a head space. Similarly, a head space may or may not be present in the first cell culture chamber. Alternatively, the spheroid-guided geometry may or may not be present in the first cell culture chamber. One skilled in the art will recognize that many combinations of these features may be desirable, depending on the user's cell culture requirements.

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

In some embodiments, one or more wells are configured based, at least in part, on their defined size and shape to grow a single spheroid of defined size. Spheroids can expand to the size limits imposed by the geometry of the wells in which they are cultured. For example, each well may contain a microwell or cell culture volume to allow spheroids to grow to a specific diameter. In other words, the geometric dimensions of the microwell or cell culture volume can limit spheroid growth such that the spheroid diameter reaches and stays at a maximum value. The production of spheroids of consistent size can lead to tissue-like, non-expandable spheroids that can be ideal for improving the reproducibility of assay results. Consistent production of spheroids can be the result of volumes of various shapes and dimensions (eg, microwells or cell culture volumes) defined by the interior of one or more wells. For example, the microwell or cell culture volume may range from about 100 micrometers to about 700 micrometers, such as from about 200 micrometers to 500 micrometers, or any range within the foregoing values (e.g., 100 micrometers to 200 micrometers, 100 micrometers, micrometers to 500 micrometers, or 200 micrometers to 700 micrometers). The microwell or cell culture volume can have a depth from about 50 micrometers to about 700 micrometers, such as from about 100 micrometers to 500 micrometers, or any range therein.

19 is a side view illustrating an additional embodiment of a cell culture device comprising a porous membrane support. 20 is a side view illustrating an additional embodiment of a cell culture device comprising a porous membrane support illustrating co-cultivation of cells.

Referring to FIG. 21 , a specific example of a cell culture apparatus 1400 having a plurality of stacked cell culture compartments 1410A, 1410B, and 1410C is shown. Each cell culture compartment may include a substrate having an array of microwells described herein. Apparatus 1400 includes a packed manifold 1430 having an opening 1435 through which cell culture medium may be introduced or removed. Filled manifold 1430 includes a plurality of openings (not shown). Each cell culture compartment 1410A, 1410B, 1410C has at least one opening (not shown) in fluid communication with an opening of the manifold 1430 so that the cell culture medium introduced through the hole 1435 passes through the cell culture compartment ( 1410A, 1410B, 1410C). The aperture 1435 may be covered with a cap (not shown), or the like, when the device 1400 is oriented into a cell culture position. Apparatus 1400 also optionally includes a vent manifold 1420 defining an opening 1425 through which air, metabolic gases, and the like may flow. Vent manifold 1420 includes a plurality of openings (not shown). Each of the cell culture compartments 1410A, 1410B, 1410C has gas and an opening in the manifold 1420 such that cell culture metabolic gases can be exchanged through the opening 1425 between the interior of the cell culture chamber and the exterior of the device 1400. It has at least one communicating opening (not shown). Aperture 1425 may be covered with a vent cap (not shown), filter (not shown), or the like when device 1400 is oriented in a cell culture position.

Referring to FIG. 22 , a cross-sectional view of a cell culture device 1400 , which may be the type of device shown in FIG. 21 , is shown. Apparatus 1400 has a plurality of stacked cell culture compartments 1410A, 1410B, 1410C, each having a substrate 1110 defining a structured surface having an array of gas permeable wells, as described above. In the embodiment shown in FIG. 22 , each compartment (eg, 1410B, 1410C), except for the topmost compartment 1410A in the stack, has an upper surface defined by the structured second major surface of the substrate 1110 of the adjacent compartment. (1450). For example, the second major surface of substrate 1110 in compartment 1410B serves as the top inner surface of compartment 1410C. Thus, the interiors of adjacent compartments are in gas communication with each other via a common substrate 1110 forming the lower structured/upper surface. Top-top section 1410 has an upper interior surface formed by top 1450, which may be a plate.

On the interior of a compartment (eg, compartment 1410A, 1410B, 1410C) is an array of microwells (eg, array 100 shown in FIG. 9 ), on the outer second major surface of the substrate of the compartment. It is defined by a substrate having a top surface defined by , and one or more sidewalls 1440 . One or more sidewalls 1440 have vent openings in communication with a vent column 1429 defined by manifold 1420, which communicates with the exterior of the device through one or more openings 1425, 1426 defined by manifold 1420. (1442). Vent opening 1442 defines the maximum volume of cell culture medium 1300 that can exist inside the cell culture chamber when the device is in a cell culture orientation (as shown in FIG. 22 ). The volume of cell culture medium within the compartment may be less than the maximum. Vent opening 1442 also defines a minimum headspace volume inside the cell culture compartment. The headspace volume inside the compartment may exceed the minimum headspace volume (if the cell culture medium volume is less than the maximum medium volume).

Thus, cells cultured in a well of a structured surface defined by a substrate 1110 of a chamber (eg, chamber 1410B) above an adjacent chamber (eg, chamber 1410C) may form a gas permeable well. Through this, gas communicates with the head space 1441 of the adjacent chamber 1410C. The head space 1441 is in a communication column 1429 defined by a manifold that communicates with the outside of the device through one or more openings 1425 and 1426.

An optional filter 1427 may be incorporated into aperture 1425 , aperture 1425 or cap 1422 to expel metabolic gases. Having a vent at the bottom of the device can be advantageous, in some embodiments. For example, the metabolic waste gas carbon dioxide is denser than atmosphere and tends to form a gradient with the highest concentration at the bottom in a culture device without a bottom vent. Thus, the presence of a vent on the bottom of the device (eg, formed by vent column 1429 and aperture 1426) can facilitate the movement of waste carbon dioxide from the device.

Referring to FIG. 23 , a cross-sectional view of a cell culture device 1400 , which may be of the type of device shown in FIG. 21 and may be part of the device shown in FIG. 22 , is shown. If each reference number in FIG. 23 is not explicitly discussed, reference is made to the discussion of similarly numbered elements described above with respect to FIG. 22 . In the illustrated embodiment, the interior of a compartment (eg, compartment 1410A, 1410B, 1410C) includes an interior structured surface of substrate 1110, a top surface defined by an exterior surface of the substrate of the compartment, and one or more It is bounded by sidewalls 1440 . One or more sidewalls 1440 communicate with the exterior of the device through an opening 1435 defined by the manifold 1430, which may be covered by a cap 1432 when the device is in a cell culture orientation. It has an opening 1443 in communication with a column 1439 defined by a manifold 1430. Cell culture medium 1300 may be introduced into or removed from the cell culture compartment (e.g., compartments 1410A, 1410B, 1410C) through column 1439 and aperture 1435 through manipulation of the device. there is.

Referring to FIGS. 24A and 24B , a schematic bottom view 24a and a schematic perspective view 24b show a cell culture compartment (e.g., as shown in FIGS. 21-23) when a plurality of trays are stacked on top of each other. trays 1415 that can serve to form compartments 1410A, 1410B, 1410C) such as bars. Tray 1415 includes sidewalls 1440A, 1440B, 1440C, 1440D extending from substrate 1110 defining a substrate having an array of microwells. In some embodiments, the tray includes a single sheathing sidewall (not shown). Part height wall 1472 connects to sidewall 1440A and sidewall 1440D. Part height wall 1472 and sidewalls 1440A, 1440D surround and define vent opening 1442 . When the cell culture device is assembled from stacked trays, metabolic gases within the interior of the chamber formed by the trays 1415 can flow over the partial walls 1472 and through the apertures 1442 . Vent opening 1442, portion wall 1472, and associated sidewalls of stacked trays 1415 can form at least a portion of a vent column (eg, vent column 1429 shown in FIG. 22).

Tray 1415 also includes part height walls 1473 connected to sidewalls 1440A and 1440B. Part height wall 1473 and sidewalls 1440A, 1440B surround and define fill opening 1443 . When the cell culture device is assembled from stacked trays, the culture medium is introduced from the inside of the chamber formed by the trays 1415 through the filling opening 1443 and over the partial wall 1473 through manipulation of the assembled device, or can be removed Packed openings 1443, portion walls 1473, and associated sidewalls of stack tray 1415 may form at least a portion of a packed column (eg, packed column 1439 shown in FIG. 23 ).

The height of portion walls 1473 defines the maximum height and volume of cell culture medium that can be contained within the cell culture compartment formed by tray 1415 . The top of partial wall 1473 can be at any suitable distance from substrate 1110 forming a structured surface. In some embodiments, the distance is greater than or equal to 5 mm, such as 6, 7, 8, 9, 10, 12, 15, or 20 mm, including any two ranges in between the aforementioned values. Since the gas permeable wells of the structured surface of the cell culture device assembled from these trays communicate with the outside of the device, the height of the cell culture medium above the cells can be greater than that available in conventional cell culture devices, where Gas exchange occurs primarily through the cell culture medium.

The distance from the top of the partial wall 1472 to the substrate 1110 may be greater than or equal to the distance from the top of the partial wall 1473 to the substrate 1110 . Thus, if the compartment is overfilled with culture medium, the excess medium will drain through the fill opening 1443 in addition to the vent opening 1442. In embodiments that use a lower filter (such as filter 1427 of FIG. 22 ), the lower filter will not be contaminated with media. Of course, proper handling of the assembled device must also prevent contamination of the bottom filter with the medium.

Referring to FIG. 25 , a schematic side view of an embodiment of a cell culture device 1400 having a plurality of stacked cell culture compartments 1410A, 1410B, 1410C is shown. Each compartment may include a substrate 1110 defining a structured surface as described above. Apparatus 1400 includes a spacer 1500 positioned adjacent to a substrate 1110 forming a structured surface and outside of the chamber to provide a pathway for air flow, which pathway is referred to herein as a "conduit". space" (tracheal space) (eg, conduit spaces 1460A, 1460B, 1460C). Because the structured surfaces define gas permeable wells in which cells can be cultured, metabolic gases can be exchanged out of the device 1400 through the wells into the conduit spaces 1460A, 1460B, 1460C defined by the spacers 1500. can Apparatus 1400 also includes a manifold 1430 defining an opening through which cell culture medium may be introduced or removed. Manifold 1430 includes a plurality of openings (not shown). Each cell culture compartment 1410A, 1410B, 1410C is in fluid communication with an opening in the manifold 1430 to allow cell culture medium introduced through the manifold 1430 to flow to the cell culture compartment 1410A, 1410B, 1410C. It has at least one opening (not shown).

In FIG. 25 , the lower portion of the cell culture device 1400 includes a plate 1510 on which the spacer 1500 is disposed. In an embodiment, plate 1510 and spacer 1500 are a single piece, such as a molded part. Such plates may form the top surface of other cell culture compartments (eg, compartments 1410A, 1410B, 1410C). For each compartment, one or more sidewalls 1440 extend from the substrate 1100 defining the structured surface to the upper surface, which may be formed as a plate with spacers. An opening (not shown) communicating with an opening (not shown) of port 1430 may be defined by a sidewall.

Stacked cell culture trays or chambers may be assembled in any suitable manner. For example, these components may be joined using welding techniques (eg, thermal, laser, long IR or ultrasonic welding, etc.), adhesion, solvent-bonding, or the like.

In some embodiments, the structured surface is connected to the bag. Bags suitable for cell culture may be formed from the film by heat sealing, laser welding, application of an adhesive, or any other method known in the art of inflatable bag manufacture. The walls of the bag, or portions thereof, may have a thickness that permits efficient movement of gas across the walls. It will be appreciated that the desired thickness may vary depending on the material from which the wall is formed. As an example, the wall or film forming the wall may be about 0.02 millimeters to 0.8 millimeters thick. The bag may be made of any material suitable for culturing cells. In various embodiments, the bag is formed of an optically transparent material, allowing visual inspection of the cells cultured in the bag. Examples of optically clear, gas permeable materials that may be used to form the bag 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 devices described herein can be used to culture cells within wells of a structured surface. As noted above, the cell culture medium in the device may be 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 of a well or nadir) is about 5 mm or greater. In some embodiments, the height of the cell culture medium in the device above the cells (eg, above the top of the well or nadir) is about 6 mm or greater, about 7 mm or greater, about 8 mm or greater, about 9 mm or greater, or about 10 mm or greater.

Because of the gas permeability of the wells, this level of cell culture medium can be used to keep the cells healthy. Thus, cells cultured in the device described herein can be cultured for a long time with a cell culture medium at this height. For example, cells can be continuously cultured at this medium level for 24 hours or longer, 48 hours or longer, 72 hours or longer, 96 hours or longer, or until the medium is changed.

In embodiments where the well is non-adherent to the cells, the cells can be harvested by inverting the device and allowing gravity to displace the cells from the well.

In some embodiments, a porous membrane is disposed in a cell culture device as described herein to support the growth of a second cell-type (or to support the growth of additional cells of the same type) within the same device, but in the device isolated to a first cell type cultured on a structured surface in gas communication with the outside. In some embodiments, stem cells are cultured on a structured surface with gas permeable wells in communication with the exterior of the device, and feeder cells are cultured on a porous membrane. Of course, any other desired combination of cells and compartmentalization may be used with this device.

A porous membrane may be disposed within the housing of the cell culture device to partition the housing into two growth chambers. Preferably, the permeable membrane restricts cell movement through the membrane but allows passage of biomolecules. Examples of materials that can be used to form the porous membrane include track-etched membranes or woven or non-woven porous materials. The material of the porous membrane may be treated or coated to make it more adherent to cells or more non-adherent. Treatment can be accomplished by a number of methods known in the art including plasma discharge, corona discharge, gas plasma discharge, ion bombardment, ionizing radiation, and high intensity UV light. The coating may be applied by any suitable method known in the art including printing, spraying, condensation, radiant energy, ionization techniques or dipping. Coatings can provide covalent or non-covalent binding sites. These sites can be used to attach moieties such as cell culture components (eg, proteins that promote growth or adhesion). Moreover, coatings can also be used to enhance adhesion of cells (eg, polylysine). Optionally, a cell non-adhesive coating described above may be used to prevent or inhibit cell binding. In some embodiments, a porous membrane can be fabricated to have 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. However, in this case, the porous membrane material is formed to have a structured surface.

Gas permeable wells of the structured surface (eg, as described above) allow control of the oxygen tension by controlling the gas concentration in the culture device in which the device is positioned to allow cell growth. Permeable membrane supports provide a method of physically separating different cell populations while allowing the delivery of biologically active components.

The porous membrane may be attached to the device's housing (eg, sidewall, etc.) in any suitable manner. For example, a porous membrane may be incorporated into a device in a manner similar to the incorporation of a substrate forming a substrate defining a structured surface having a plurality of gas permeable wells, as described above.

In the embodiment shown in FIG. 26 , cell culture device 3100 is a 96-well multiwell plate with wells 3115 in plate 3111 surrounded by frame 3113 . However, as noted above, a cell culture device may have any suitable 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 contains a substrate having an array of microwells and, as described above, provides a location to form a spheroid. Almost any type of cell culture device having a well used to culture cells can, in some embodiments, form the entire volume of the well, or in some embodiments, form a cell culture sub-volume of the well. It can be designed using a substrate with an array of microwells on it. In some embodiments, a cell culture device in which a microwell design can be implemented can be a multi-well plate, eg, a 96-well multiwell plate, a 384-well multiwell plate, a 1536-well multiwell plate, etc. there is. In some embodiments, at least one surface of the multiwell plate is gas permeable.

The ability of the spheroids formed within each well of a plurality of wells to maintain a constant size across the plurality of wells, as well as the ability to aggregate cells in such a way that the plurality of wells form spheroids, can be achieved in any suitable manner. . For example, plurality of wells 3115 can include an array of microwells structured and arranged similarly to the microwells shown in FIGS. grow up

In some embodiments, one or more wells are configured, based at least in part on their defined size and shape, to grow a single spheroid of defined size. Spheroids can expand to the size limits imposed by the geometry of the wells in which they are cultured. For example, each well may contain a microwell or cell culture volume that allows spheroids to grow to a specific diameter. In other words, the geometric dimensions of the microwell or cell culture volume can limit spheroid growth such that the spheroid diameter reaches and remains at its maximum value. The production of spheroids of consistent size can lead to tissue-like, non-expandable spheroids that can be ideal for improving the reproducibility of assay results. Consistent production of spheroids can be the result of varying shapes and dimensions volumes (eg, microwells or cell culture volumes) defined by the interior of one or more wells. For example, the microwell or cell culture volume may range from about 100 micrometers to about 700 micrometers, such as from about 200 micrometers to 500 micrometers, or any range within the foregoing values (e.g., 100 micrometers to 200 micrometers, 100 micrometers to 500 micrometers, or between 200 micrometers and 700 micrometers). A microwell or cell culture volume can have a depth from about 50 micrometers to about 700 micrometers, such as from about 100 micrometers to 500 micrometers, or any range therein.

Each of the plurality of wells described herein can assist cells deposited therein to form spheroids. In addition, each of the plurality of wells, for example, 500 micrometers or less, 400 micrometers or less, 300 micrometers or less, 250 micrometers or less, 150 micrometers or less, or any range within the above range (eg, 150 to 250, 150 to 300, 150 to 400, 150 to 500, 250 to 300, 250 to 400, etc.) to limit or constrain the diameter of each of the spheroids. In some embodiments, each of the plurality of wells has a diameter that differs from the average diameter of all spheroids grown in the plurality of wells, e.g., about 20% or less, 15% or less, 10% or less, 5% or less, 2% or less. Or form a spheroid limited to any range within the above range.

In the embodiments shown in FIGS. 27-29 , an array of microwells 3115 is formed in a substrate 3110 . Each of the plurality of wells 3115 can have an upper opening 3118 , a lower surface 3112 , and a sidewall surface 3120 extending from the upper opening 3118 to the lower surface 3112 . Additionally, sidewall surface 3120 can define a pen tip region 3116 (see FIG. 29 ) between upper opening 3118 and lower surface 3112, which constricted region at the bottom of the well is a pen tip region. It is so named because it resembles the tip of The Syck pen tip region is a spheroid-derived geometry.

The upper opening 3118 of each of the plurality of wells 3115 can be used as an opening to seed each of the plurality of wells 3115 with cells. Upper opening 3118 can have a variety of different shapes and sizes. For example, upper opening 3118 may be defined by a shape such as round, oval, square, rectangular, hexagonal, quadrilateral, or the like. Upper opening 3118 may also be defined by a diameter dimension (eg, diameter, width, etc., depending on shape). The diameter dimension of upper aperture 3118 may be defined as the distance across the upper aperture at its widest point. The diameter dimension of upper opening 3118 is, for example, 300 micrometers or greater, 500 micrometers or greater, 800 micrometers or greater, 1000 micrometers or greater, 1500 micrometers or greater, 2000 micrometers or greater, or 7000 micrometers or less, 6000 micrometers or less, 4000 micrometers or less. , 2500 micrometers or less, 1700 micrometers or less, 1200 micrometers or less, or any range within the aforementioned values.

The lower surface 3112 of each of the plurality of wells 3115 may be conducive to allowing cells to be cultured on or thereon. Lower surface 3112 can have a variety of different shapes and sizes. For example, lower surface 3112 can be round, hemispherical, flat, conical, or the like. As another example, lower surface 3112 can also be defined by a shape such as circle, oval, square, rectangle, hexagon, quadrilateral, or the like. 27-29, lower surface 3112 is flat and defined by a diameter dimension. The diameter dimension of the lower surface is, for example, 0 micrometers or greater, 50 micrometers or greater, 75 micrometers or greater, 100 micrometers or greater, 200 micrometers or greater, 275 micrometers or greater, etc., or 700 micrometers or less, 500 micrometers or less, 400 micrometers or less, 300 micrometers or less micrometers or less, 250 micrometers or less, 150 micrometers or less, or any range within the foregoing values. For lower surface 3112 having a rounded lower or similar surface (eg, hemispherical, conical, etc.), with the nadir located at its lowest point, the diameter dimension of lower surface 112 is considered zero. In some embodiments, a diameter dimension of upper opening 3118 is greater than a diameter dimension of lower surface 3112. In other embodiments, the diameter dimension of upper opening 3118 is the same as the diameter dimension of lower surface 3112.

In some embodiments, the lower surface 3112 of each of the plurality of wells 3115 can be uniformly composed of a substrate having a microarray of wells (see FIG. 29 ). In other embodiments, lower surface 3112 may be made of a material different from that used to form substrate 3110 . Various methods of fabricating the plurality of wells will be further described below. Bottom surface 3112 or sidewall surface 3120 may be gas permeable to help provide oxygen to cells or spheroids 3130 cultured within well 3115 . In some embodiments, the substrate 3150 defining the lower surface 3112 can be a gas permeable substrate. In some embodiments, substrate 3150 can include a gas permeable film. The permeability of lower surface 3112 to the outside will depend in part on the material of lower surface 3112 and the thickness of lower surface 3112 . For example, gas permeability of wells is disclosed in US Provisional Patent Application No. 62/072,088, filed on October 29, 2014 entitled "GAS PERMEABLE CULTURE FLASK", the entire content of which is in line with this disclosure. Insofar as they do not conflict, they are incorporated herein by reference.

Pen tip region 3116 (see FIG. 29 ) can be defined by sidewall surface 3120 between upper opening 3118 and lower surface 3112 . The location of the pen tip region 3116 may be defined by other components of the well. For example, pen tip region 3116 can be defined by a diameter dimension 3144 across sidewall surface 3120 . A diameter dimension 3144 of the pen tip region 3116 can be defined as the distance across the sidewall surface 3120 at the pen tip region 3116 . Pen tip zone 3116 is, for example, 50 micrometers or greater, 100 micrometers or greater, 200 micrometers or greater, 300 micrometers or greater, 400 micrometers or greater, 550 micrometers or greater, or 800 micrometers or less, 700 micrometers or less, 600 micrometers or less, 500 micrometers or less. It may be defined by diameter dimension 3144 of micrometers or less, 450 micrometers or less, 350 micrometers or less, etc. or any range therein. Pen tip zone 3116 may also be, for example, greater than 50 micrometers, greater than 100 micrometers, greater than 150 micrometers, greater than 250 micrometers, greater than 350 micrometers, greater than 450 micrometers, etc., or less than 800 micrometers, less than 700 micrometers, less than 600 micrometers. , 500 micrometers or less, 400 micrometers or less, 300 micrometers or less, etc. or any range therein may be defined by the height 3142 from the lower surface 3112 . Height 3142 may be measured from the lowest point of lower surface 3112 . In this embodiment, the total volume of well 3115 is the cell culture volume 3140.

The diameter dimension of the upper opening 3118 can be greater than or equal to the diameter dimension 3144 of the pen tip region 3116. A diameter dimension 3144 of the pen tip region 3116 may be greater than or equal to a diameter dimension of the lower surface 3112 . Further, the diameter dimension of the lower surface 3112 can be less than or equal to the diameter dimension 3144 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. may be described as In some embodiments, top opening 3118 can be pen tip region 3116.

Each sidewall surface 3120 of the plurality of wells 3120 extends from an upper opening 3118 to a lower surface 3112 . Sidewall surface 3120 can include an upper sidewall surface 3124 and a lower sidewall surface 3122 . An upper sidewall surface 3124 can be defined between the upper opening 3118 and the pen tip region 3116. A lower sidewall surface 3122 can be defined between the pen tip region 3116 and the lower surface 3112 . In some embodiments, the sidewall surface 3120 of each of the plurality of wells 3115 can be defined as a cell non-adherent surface. The cell non-adherent surface facilitates the growth of cells into spheroids 3130 in the cell culture volume 3140 as described above. The cell non-adherent top sidewall surface 3124 can facilitate establishment of seeded cells into the cell culture volume 3140 . Regardless of whether upper sidewall surface 3124 is cell non-adherent, in some embodiments, upper sidewall surface 3124 is structured such that cells seeded in wells settle into pen tip region 3116, A cell culture volume 3140 forms as a result of gravity.

In some embodiments, upper sidewall surface 3124 and lower sidewall surface 3122 can be defined by, for example, a parabolic, conical, stepped, angular, curved, etc. shape. The upper and lower sidewall surfaces 3124 and 3122 may have the same or different shapes. In some embodiments, sidewall surface 3120 can have an inflection point 3121 at a location where upper and lower sidewall surfaces 3124 and 3122 meet (eg, as shown in FIG. 29 ). In other embodiments, the sidewall surface 3120 can have a continuous slope at the inflection point 3121 where the upper and lower sidewall surfaces 3124 and 3122 meet.

In some embodiments, a portion of the sidewall 3120 adjacent to the lower surface 3112 may be perpendicular to the lower surface 3112 or at an angle to the lower surface 3112 . The portion of sidewall 3120 adjacent to lower surface 3112 may be described as lower sidewall surface 3122 . The angle at which a portion of the sidewall 3120 intersects the lower surface 3112 is, for example, greater than or equal to 90 degrees, greater than or equal to 92 degrees, greater than or equal to 95 degrees, greater than or equal to 100 degrees, etc., or less than or equal to 110 degrees, less than or equal to 105 degrees, or 102 degrees. may be defined for lower surface 3112 as less than or equal to 97 degrees, etc., or any range within the foregoing values. In some embodiments, a diametric dimension across sidewall surface 3120 may be described as increasing from lower surface 3112 toward upper opening 3118. The sidewall geometry may be any geometry sufficient to allow cells to settle into each of the plurality of wells 3115.

With respect to lower surface 3112 not having a flat surface, the angle of sidewall 3120 is considered relative to an imaginary plane tangent to the nadir of lower surface 3112 . In other embodiments, the imaginary plane can also be defined as being coplanar with the top opening 3118 whether or not the imaginary plane is tangential to the nadir.

A combination of bottom surface 3112 , pen tip region 3116 , and a portion of sidewall surface 3120 may define a cell culture volume 3140 . The portion of the sidewall surface 3120 that defines the cell culture volume 3140 may also be described as a lower sidewall surface 3122. Cells are not limited to being cultured only in the cell culture volume 3140. However, cells deposited within each of the plurality of wells 3115 may aggregate in the cell culture volume 3140 to form and grow spheroids 3130 . Additionally, the dimensions of the spheroid 3130 can be a result of the shape and size of the cell culture volume 3140 . For example, the cell culture volume 3140 of each of the plurality of wells 3115 is such that the spheroid 3130 has a thickness of, for example, about 500 micrometers or less, 400 micrometers or less, 300 micrometers or less, 250 micrometers or less, 150 micrometers or less. configured to grow to a diameter that is within micrometers or less, etc., or any range within the foregoing values. In some embodiments, each cell culture volume 3140 of the plurality of wells 3115 has a diameter different from the average diameter of all spheroids 3130 grown in the plurality of wells 3115, for example about 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, or any range within the foregoing.

The combination of upper opening 3118 , pen tip region 3116 and a portion of sidewall surface 3120 can define a second volume 3145 . The portion of sidewall surface 3120 that defines second volume 3145 may also be described as upper sidewall surface 3124 . Second volume 3145 may exceed cell culture volume 3140 . For example, the second volume 3145 is at least about 100%, at least 200%, at least 500%, at least 1,000%, at least 10,000%, at least 100,000%, at least 200,000% of the cell culture volume 3140 or any of the foregoing values. It can be any range within. As an example, a 96-well plate can have a cell culture volume defined by a volume of 0.1 microliters and a second volume defined by a volume of 200 microliters, resulting in a second volume that exceeds the cell culture volume by a factor of 2,000.

One embodiment of a cell culture device 3100 is illustrated in FIG. 27 . As shown in FIG. 27 , the sidewall surface 3120 of each of the plurality of wells 3115 tapers from an upper opening 3118 to a lower surface 3112 . Specifically, sidewall surface 3120 extends from upper opening 3118 in a direction substantially perpendicular to upper opening 3118, but as sidewall surface 3120 extends toward lower surface 3112, sidewall surface 3120 It extends at a slight angle reducing the diameter dimension across the . At a point 3123 along sidewall surface 3120 between upper opening 3118 and pen tip region 3116, the angle of sidewall surface 3120 is such that sidewall surface 3120 faces lower surface 3112. As it extends, it changes to further reduce the diameter dimension across the sidewall surface 3120. In the pen tip region 3116, the angle of the sidewall surface 3120 changes once again and extends towards the lower surface 3112. This last portion of sidewall surface 3120 is sometimes described as lower sidewall surface 3122 . As shown in FIG. 27 , in an embodiment, the lower sidewall surface 3122 is slightly angled from normal to the lower surface 3112 and the diameter dimension across the sidewall surface 3120 is such that the sidewall surface 3120 is decreases as it extends toward the lower surface 3112. Spheroid 3130 is shown positioned within lower sidewall surface 3122 and opposite sidewall surface 3112 (ie, pen tip region 3116 ). Lower sidewall surface 3122 defines or can limit the size to which spheroid 3130 can grow.

One embodiment of a cell culture device 3100 is illustrated in FIG. 28 . As shown in FIG. 28 , the sidewall surface 3120 of each of the plurality of wells 3115 tapers from an upper opening 3118 to a lower surface 3112 . Specifically, sidewall surface 3120 initially extends from upper aperture 3118 at an angle perpendicular to upper aperture 3118, and then extends along a parabolic path toward pen tip region 3116. In the pen tip region 3116, the angle of the sidewall surface 3120 changes to extend toward the lower surface 3112. This last portion of sidewall surface 3120 is sometimes described as lower sidewall surface 3122 . As shown in FIG. 29 , lower sidewall surface 3122 is slightly angled from normal to lower surface 3112, and the diameter dimension across sidewall surface 3120 is such that sidewall surface 3120 has lower surface 3112 ) decreases as it extends toward Spheroid 3130 is shown positioned within lower sidewall surface 3122 and opposing lower surface 3112 at pen tip region 3116 . Lower sidewall surface 3122 defines or may limit the size to which spheroid 3130 can grow. The pen tip area is a spheroid derived geometry.

Referring to FIG. 30 , in some embodiments, a cell culture device 3650 may include a bottom plate 3610 and one or more sidewalls 3620, as shown in FIG. 30 . Bottom plate 3610 can define a major surface 3611 and one or more sidewalls 3620 can extend from bottom plate 3610 . The lower plate 3610 may be formed, in whole or in part, from a substrate having an array of microwells 3615. 30 illustrates that the bottom plate can have an array of microwell 3615 arrays. That is, each of the regions identified as an array of microwells 3615 shown in FIG. 30 may contain an array of much smaller microwells. In an embodiment, the cell culture device 3650 may also include a plurality of wells 3615 formed in the major surface 3611 of the lower plate 3610 . Each well of the plurality of arrays of microwells 3615 may define a microwell or cell culture volume, as described above, that promotes or induces growth of spheroids. A major surface 3611 of the bottom plate 3610 and one or more sidewalls 3620 define a reservoir volume. The reservoir plates described herein allow for the addition of culture medium in excess of that typically used to fill individual shallow wells of microwell plates and allow cells cultured in other wells to be in fluid communication.

In some embodiments, one or more of the sidewalls 3620 may extend farther (eg, sidewall height) from the lower plate 3610 than some currently available cell culture devices, such that the reservoir has a larger than normal volume of media. It makes it possible to retain the positive medium. The opportunity for a larger capacity for the reservoir allows 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 frequently as spheroids growing in standard microplate wells. As shown in Figure 30, nutrients and metabolites can be exchanged throughout the cell culture medium because the cell culture medium within the reservoir communicates with all wells within the reservoir.

In some embodiments, cell culture assembly 3600 can include a cell culture device 3650 and a fluid permeable mesh 3670. A fluid permeable mesh 3670 may be placed on top of the wells 3615 after cells have been seeded into the wells. Cell culture medium in common communication among the plurality of wells 3615 can be removed and replaced during a manual batch feeding process without disturbing the cells within the wells. Cell culture medium exchange without disrupting or losing spheroids can be difficult because the cells, in some embodiments, may be non-adherent to the surface of the well. However, the use of mesh 3670 as described above may alleviate this difficulty. For example, a combination of a cell culture device and a fluid permeable mesh can be described in U.S. Provisional Patent Application No. 62/072,103, filed on October 29, 2014 entitled "RESERVOIR Spheroid Plate", wherein the The patent application is incorporated herein by reference in its entirety to the extent that it does not conflict with this disclosure.

It will be appreciated that the wells 3615 of the cell culture device 3650 described herein may be of any size, shape or configuration. In some embodiments, the wells are formed from hexagonal dense well structures as shown in FIG. 17 . Reservoir plate devices having structured surfaces with densely packed low volume wells can be particularly advantageous since the low volume wells require frequent exchange of cell culture medium without added reservoir volume.

Reservoir plates described herein allow for the addition of culture medium in excess of that typically used to fill individual shallow wells of microwell plates, for example, and allow cultured cells in other wells to be in fluid communication. do.

As shown in FIG. 31 , cell culture device 4100 can include a bottom plate 4110 and one or more sidewalls 4120 . The bottom plate can define a major surface, and one or more sidewalls 4120 can extend from the bottom plate. A combination of a bottom plate and one or more sidewalls may define a reservoir. The cell culture device may also include, in whole or in part, a substrate having an array of microwells 4115 formed on a major surface of the bottom plate. Each well of the plurality of wells in the microwell array may define a top opening and nadir. The upper opening may be co-planar with the major surface and the nadir may be located below the major surface, ie the nadir may be located opposite the direction in which one or more sidewalls extend from the lower plate. A top plate (not shown) can be placed over the reservoir as desired while culturing the cells.

In some embodiments, one or more of the sidewalls may extend farther from the lower plate than would normally be possible, thus allowing the reservoir to hold a larger than normal media volume. The opportunity for a larger capacity 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, the spheroids do not need to be supplied with cell culture medium as frequently as spheroids growing in standard microplate wells. As shown in Figure 31, since the cell culture medium in the reservoir is in communication with all wells in the reservoir, nutrients and metabolites can be exchanged throughout the cell culture medium.

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

In some embodiments (eg, as shown in FIG. 32 ), frame 4560 can be connected to mesh 4570 , as shown. The frame 4560 can be configured to hold the mesh 4570 in place over the first well 4515 of the wells. In some embodiments, the mesh 4570 is configured to be disposed over the top edge of the sidewall 4120 of the device 4100. Frame 4560 may engage one or more sidewalls 4120 via an interference fit, snap fit, or any other suitable mechanism for retaining the mesh on the major surface of plate 4110. can In some embodiments, the user manually holds the frame 4560 in place such that the mesh is held on the major surface of the plate 4110 over the wells 4115.

Fluid permeable mesh 4570 can be formed of any suitable material. In some embodiments, the fluid permeable mesh defines pores. The pores may 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 of a size small enough to prevent the passage of spheroids through the mesh.

In some embodiments, meshes are described, for example, in commonly-assigned US Provisional Patent Application No. 62/072094, the entire contents of which are incorporated herein by reference to the extent it does not conflict with this disclosure.

In some embodiments, instead of manually replacing the cell culture medium, a reservoir plate as described herein may be fabricated as a perfusion device through which cell culture medium can flow across the reservoir over the major surface of the well. can

For example, referring to FIG. 33 , a cell culture device 4100 as described and described in connection with FIG. 31 has one or more sidewalls defining an inlet 4140 and one or more sidewalls defining an outlet 4145 . can be modified to Cell culture fluid may be perfused across the reservoir from the inlet to the outlet. The form factor of the device shown in FIG. 33 can be an open top form factor or a closed top. If the form factor is open top, an insert comprising a frame and mesh as discussed in connection with FIG. 2 may be used if the high perfusion rate is capable of removing cells, such as spheroids, from the well. (4115) can be used to retain cells.

A cell culture device as described herein may be constructed in any suitable manner. In various embodiments, a method of fabricating a cellular device includes molding a polymeric material, or any other suitable material as described herein, to form a cell culture device. The polymeric material may define a plurality of wells of the cell culture device. Each of the plurality of wells may define an upper opening, a lower surface, and a sidewall surface extending from the upper opening to the lower surface. The sidewall surface may also define a pen tip area between the upper opening and the lower surface. The polymeric material may be poured into a mold having pins such that the polymeric material molded around the pins has the characteristics of multiple wells as described herein.

In some embodiments, a polymeric material is overmolded onto a substrate to form a cell culture device. The substrate defines the lower surface, and the combination of polymeric material and substrate defines a plurality of wells. The polymeric material may be poured into a mold having pins such that the polymeric material molded around the pins has the characteristics of multiple wells as described herein.

In some embodiments, regardless of how the cell culture device described herein is fabricated, the sidewall surface of each of the plurality of wells may be coated with a cell non-adherent material, as further described herein.

In some embodiments, the well is part of or includes various features added to the sidewall. These substrate features can be injection molded directly or embossed onto a formed substrate. The featured material can be a polymer, polymer blend, co-polymer, 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 formed of any suitable material. Preferably, materials intended to contact the cells or the culture medium are compatible with the cells and the medium. Typically, cell culture components are formed from polymeric materials. Examples of suitable polymeric materials include polystyrene, polymethylmethacrylate, polyvinylchloride, polycarbonate, polysulfone, polystyrene copolymers, fluoropolymers, polyesters, polyamides, polystyrene butadiene copolymers, fully hydrated styrene polymers, poly carbonate PDMS copolymers, and polyolefins such as polyethylene, polypropylene, polymethyl pentene, polypropylene copolymers and cyclic olefin copolymers, and the like.

In an embodiment, the inner surface of the well is non-adherent to cells. The wells may be formed from a non-adherent material or may be coated with a non-adherent material to form a non-adherent surface. Examples of non-adhesive materials include perfluorinated polymers, olefins or similar polymers or mixtures thereof. Other examples include agarose, non-ionic hydrogels such as polyacrylamide, polyethers such as polyethylene oxide, and polyols such as polyvinyl alcohol, or similar materials or mixtures thereof. In some embodiments, a combination of, for example, two or more non-adherent wells, the geometry of the wells, and/or gravity induces cells cultured in the wells to self-assemble into spheroids. Some spheroids retain differentiated cell functions that exhibit a response that is rather close to in vivo than cells grown in monolayers. In embodiments where the well is non-adherent to the cells, the cells can be harvested by inverting the device allowing gravity to displace the cells from the well.

In some embodiments, surface modifications of the material are used to achieve desired properties. These modifications include modification of surface chemistry and mechanical properties using biological coatings (eg, Matrigel™, collagen, laminin, etc.) and synthetic coatings (eg, Synthemax®, silicone hydrogels, etc.) and can be utilized. Other surface modifications to the material (eg, within wells or microstructures) are within the scope herein.

A substrate having a structured surface as described herein may be assembled into a cell culture chamber or tray in any suitable manner. For example, the structured surface and one or more other components of the cell culture chamber or tray may be molded as a single part. In some embodiments, a structured surface or a portion thereof that is overmolded to form a bottom and one or more components, the structured surface is welded (e.g., thermal, laser, long IR or ultrasonic welding, etc.), adhered, solvent-bonded or the like.

In various embodiments, a cell culture system may include one or more of the cell culture device components described herein above. For example, device components may be stacked to form a cell culture system. Examples of stacked cell culture systems that may incorporate cell culture device components as described herein include, for example, (i) U.S. patent application filed on October 29, 2014 entitled "MULTILAYER CULTURE VESSEL"; Provisional Patent Application No. 62/072,015; (ii) including what is disclosed in U.S. Provisional Patent Application Serial No. 62/072,039, filed on October 29, 2014 entitled "PERFUSION BIOREACTOR PLATFORM", the entire contents of each of which do not conflict with the present disclosure; incorporated herein by reference.

The cell culture devices described herein may be used to culture cells within the wells of the device in any suitable manner. For example, a method of culturing cells includes introducing cells and cell culture medium into one or more plurality of wells of a cell culture device as described herein. The cell culture medium may be contained throughout each of the cell culture volume or the plurality of wells comprising the cell culture volume and the second volume. The method also includes culturing the cells in a medium in one or more plurality of wells. Culturing the cells in one or more plurality of wells may include forming spheroids within the one or more wells. Spheroids cultured in one or more wells can be defined, for example, to a diameter of about 500 micrometers or less, 400 micrometers or less, 300 micrometers or less, 250 micrometers or less, 150 micrometers or less, or any range within the foregoing values. there is. The diameter of one spheroid can differ from the mean diameter of all spheroids grown in the plurality of wells, for example, by about 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, or may vary by any range within the aforementioned values.

In some embodiments, the well-bottom comprises a concave arcuate surface or “cup” geometry, such as a hemispherical surface, a conical surface with a rounded bottom, and similar surface geometries, or combinations thereof. Wells (e.g., microwells) and well-bottoms may ultimately be end-to-end, terminated, or finished with spheroid "friendly" rounded or curved surfaces, concave frusto-conical relief surfaces, such as dimples, or combinations thereof. can

In certain embodiments, the sidewalls and/or portions of the bottom-well are of varying degrees of opacity/transparency for wavelengths within the visible and/or UV spectrum. For example, an opaque sidewall can be combined with a transparent microwell-bottom. The transition from an opaque portion to a transparent portion may be gradual or instantaneous.

In some embodiments, a well (eg, a microwell) includes a low-adhesion, non-adhesion, or high-adhesion coating on a portion of the well, eg, on at least one concave arcuate surface.

In some embodiments, the device further includes, for example, a well adjunct, a well extension region, or an auxiliary subchamber for receiving a pipette tip for suction. In some embodiments, a well adjunct or well extension (eg, side pocket) is an integral surface adjacent to and in fluid communication with, for example, a well (eg, microwell). In some embodiments, the well fitting has a lower portion spaced from a gas permeable, transparent lower portion of the well. The well adjunct and second lower portion of the chamber well are spaced apart from the gas-permeable, transparent lower portion, such as at a higher elevation or relative elevation. In some embodiments, the second lower part of the well accessory diverts fluid dispensed from the pipette away from the transparent lower part so as not to disturb or unbalance the spheroid.

In certain embodiments, the device further comprises a porous membrane, such as a liner or membrane insert, located within a portion of a well, within a portion of a well adjunct, or located on both a well and a well adjunct portion. The porous membrane is formed from the first intracellular material in the lower part of one or both wells near the bottom, located in the upper part of the well, located in the upper part of the well formed by the porous membrane, or located in both wells. , sequestration or separation of a second intracellular substance, such as another cell type or other cell state.

Devices and well geometries are fabricated by any suitable technique known in the art. In some embodiments, hot embossing, heat deformation, and/or injection molding methods are used to prepare micropatterned surfaces in cell-culture-compatible plastics. 12 shows a schematic diagram of the hot embossing/thermoforming process used herein. In some embodiments, a polystyrene film (or another suitable polymeric film) of a certain thickness is placed on a resistive silicone support on the thermal bed. The mold is then placed on the film with the microposts facing down. The entire assembly is pressed with a 5N load between plates preheated to 130°C for 10 minutes. After 10 minutes, the plate is cooled down to 100° C., and the micropatterned embossed/thermoformed film is removed from the assembly and incorporated into a cell culture vessel, usually as a 3D aggregation promoting surface. Other temperatures, times, pressures and materials may be within the scope herein.

To prevent cell adhesion, in some embodiments, the micropatterned surface is treated with a polymer that inhibits cell adhesion, such as poly-HEMA, pluronic, or a proprietary ULA treatment. Depending on the initial polymer film thickness and process parameters, microwelled surfaces with different underlying thicknesses are created. In some embodiments, the thickness of the polymer at the bottom of the microwell directly affects the oxygen permeability. A thin microwell bottom allows better oxygen supply to the cells located inside the microwells. The fabrication method yields a surface with highly oxygen permeable microwells.

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

In vitro 3D tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers. In some embodiments, cell culture formats with microwell patterned surfaces, as described herein, can be used for 3D printing in large quantities of uniform size, compatible with routine high-throughput drug development and preclinical research. generating a culture (eg, tumor spheroid).

In some embodiments, the culture vessels described herein are used for the formation of embryoid bodies (EBs) derived from induced pluripotent stem (iPS) cells and embryonic stem cells (ESCs) to form uniform and facile aggregates on a large scale. makes it possible In some embodiments, medium exchange is performed to allow EBs to grow continuously for several weeks. In some embodiments, the size of each aggregate is controlled as the size depends on the number of cells inoculated and the time of incubation. In some embodiments, aggregates are transferred to larger volumes of media or traditional well plates allowing analysis of spheroids. In some embodiments, multiple formed EBs provide statistically significant data from high-throughput analysis of infected targets or small molecules in a single plate. The ability to support the formation of multiple 3D cell aggregates in one culture vessel makes these vessels, eg, modified Petri dishes, applicable for the selection of EB-forming clones for cell reprogramming. In some embodiments, the vessels described herein also support the formation of 3D cell aggregates in various cell types relevant to toxicology, such as hepatocytes and embryonic stem cells. In some embodiments, microwell surface vessels are used for 3D aggregate cell culture for the purpose of protein production, for example in the field of biological manufacturing. In some embodiments, a cell culture format having a microwell patterned surface, as described herein, is a stem cell niche co-culture, particularly a clonogenic culture, a single stem A means enabling cell and in situ co-culture is provided. In combination with established staining protocols for imaging, as well as stem cell surface markers or stem cell differentiation markers, in situ co-culture and computational identification of single stem cells can be performed.

Micropatterned vessels are also used for cell banking purposes to preserve cells in a 3D format. In some embodiments, once the spheroids have grown to a certain size (usually in a transition state prior to a steady state), they are removed from the microwells and the collected spheroids are cryopreserved and stored for future use. Transitional spheroids mean that the spheroid can continuously grow in size, whereas stable spheroids mean that the spheroid does not grow in size once it reaches its intrinsic size limit. A variety of cryopreservation methods, including but not limited to dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and the like, are within scope herein.

embodiment

Example 1: substrate manufacturing

A substrate according to an embodiment is manufactured using an embossing method, as illustrated in FIG. 12 . 12 shows a high-temperature embossing/thermoforming process of microwell formation in a polymer film. A hot plate (1) is provided. The hot plate is preheated to 130°C. An embossing mold 2 is provided to reflect the desired well profile. A layer (3) of polymeric film is provided, and a silicone mat (4) is provided behind the polymeric film. The hot plate is heated and pressed against the polymer film, backed by a layer of silicon. When the hot plate is removed, the polymeric film having an array of microwells having the desired well profile embossed therein is provided.

Example 2: Cell culture

Experiments performed during the development of the embodiments described herein show, for example, that uniform diameter 3D cell culture aggregates have a diameter (D) that is about 1 to 3 times the desired diameter of the 3D cell aggregates, hemispherical round bottom and round top It is shown to be formed and cultured in an array of individual, spatially separated microwells each having a. The microwell height (H) is about 0.7 to 1.3 times the diameter of the round bottom part, and the diameter of the top opening of the microwell (D _top ) is 1.5 to 2.5 times the round bottom diameter.

Experiments are conducted in which a T25 cell culture flask prototype with microwell patterned surface is fabricated and tested in cell culture applications to demonstrate the uniformity of spheroid formation and retention/harvesting benefits of the proposed design. . 8 contains inverse replica images of T25 prototype microwell cross-resections with rounded bottom geometry. An image of spheroids formed by HT29 cells in a prototype T25 flask is shown in FIG. 10A. FIG. 10B shows spheroids harvested from flasks comparable to those grown using commercially available NUNCLON SPHERA™ low binding surface flasks, available from Nunc Nunc/Thermo Fisher, shown in FIG. 11 ; The superior performance of the wells described herein is evident in the uniformity of the spheroids produced in the embodiments compared to commercially available controls that have a low binding surface but no wells to confine and control spheroid production. The round subgeometry is a spheroid derived geometry. Experiments were performed during development of embodiments herein to demonstrate the effect of oxygen permeability of microwells on cell culture performance. 6 and 12 well plates were fabricated as described above in Example 1, with microwells of different thicknesses. 13 shows a graph showing the number of viable cells measured after growing cells in 6 well plates with substrates containing microwell arrays (as described in Example 1), in microwells with different bottom thicknesses. As shown in FIG. 13, A is 70 μm thick, B is 120 μm thick, and C is 320 μm thick. The control is TCT-treated 1 mm thick flat polystyrene. As can be seen from the results presented in FIG. 13, thinner materials (which exhibit more gas permeability), in embodiments, support more vigorous cell growth.

13 is a graph showing the number of viable cells measured after growing cells in a 6 well plate having a substrate containing an array of microwells (as described in Example 1), in microwells with different bottom thicknesses. . 14A and 14B show graphs comparing viable cell counts and cell productivity for substrates with microwell arrays compared to flat surfaces. 15 is a graph depicting total protein titers shed from MH677 cells cultured on substrates with microwell arrays compared to flat surfaces.

Cultivation of MH677 cells was performed for 7 or 9 days with medium exchange every 2 days. The results presented in FIG. 13 show the dependence of the total number of viable cells on the microwell bottom thickness. Cells cultured in oxygen permeable microwells (column A, 70 μm thick bottom) yield 82% higher viable cell counts compared to low oxygen permeability column C, 320 μm thick bottom. Overall, culture on microwell patterned surfaces yielded higher viable cell numbers (FIG. 14A) and higher productivity per cell (FIG. 14B) compared to normal flat, non-adherent surfaces (FIG. 14). do. This produces an 85% higher protein yield (FIG. 15).

Experiments are performed during development of embodiments herein to demonstrate beneficial growth of cells in 2D versus 3D culture using the cell culture devices and wells described herein. In both CHO 5/9 alpha cells (FIG. 34A) and BHK-21 cells (FIG. 34B), significantly more proteins (hm-CSF in FIG. 34A, EPO in FIG. 34B) per cm in 3D culture compared to 2D. is produced

This application and/or all publications and patents listed below are incorporated herein by reference. Various modifications, recombinations, and variations of the described features and embodiments will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although specific embodiments have been described, it is to be understood that the invention as claimed is not unduly limited to these specific embodiments. Indeed, various modifications of the described manners and embodiments that are obvious to those skilled in the art are intended to be within the scope of the following claims.

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. Roppella, Essential operating principles for tumor spheroid growth. 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 antiinflammatory 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

Claims (20)

As a cell culture substrate,
comprising an array of microwells, each microwell comprising a hole into the microwell defined by a rounded top well edge, at least one microwell sidewall and a rounded well-bottom;
Here, the array of microwells has a sinusoidal or parabolic shape defining the cell culture surface on one side and having a surface opposite the cell culture surface in communication with the outside of the cell culture substrate. , cell culture substrates. The method of claim 1,
The cell culture substrate further comprising one or more ridges, wherein the ridges are circular, prismatic, acicular-shaped or hexagonal. The method of claim 1,
The cell culture substrate further comprising one or more fissures, wherein the fissures are circular, prismatic, acicular-shaped or hexagonal. The method of claim 1,
The lower portion of the round well has a thickness of 10 to 100 μm, cell culture substrate. The method of claim 1,
The cell culture substrate of claim 1 , wherein the walls of the microwell are relatively thick near the hole into the microwell and relatively thinner at the bottom of the microwell. The method of claim 1,
Wherein the substrate comprises 2-10000 microwells per cm 2 of the surface of the cell culture substrate. The method of claim 1,
The cell culture substrate of claim 1 , wherein the at least one rounded well-bottom comprises a non-adherent surface. The method of claim 1,
The microwell sidewall is discontinuous, cell culture substrate. The method of claim 8,
The microwell sidewall is corrugated, cell culture substrate. The method of any one of claims 1-9,
Wherein the cell culture substrate comprises at least a portion of a cell culture vessel. The method of claim 10,
The cell culture substrate of claim 1 , wherein the cell culture vessel is selected from the group consisting of multi-well plates, dishes, flasks, tubes, multi-layer flasks, soft-sided flasks and bags. The method of any one of claims 1-9,
The cell culture substrate of claim 1 , wherein the microwells are configured to allow fluidic communication between at least one of the microwells and a single liquid reservoir. The method of claim 10,
The cell culture substrate of claim 1, wherein the cell culture vessel further comprises a mesh positioned on top of the array of microwells. The method of any one of claims 1-9,
A cell culture substrate, wherein at least the round well-bottom comprises a gas permeable material. The method of any one of claims 1-9,
A cell culture substrate, wherein each microwell has a sinusoidal well geometry in cross section. The method of any one of claims 1-9,
the hole into the microwell has an effective diameter (D _top ), the round well-bottom has a nadir, and each microwell has one or more sidewalls; The at least one sidewall has a height (H) extending from the nadir of the round well-bottom to the hole into the microwell, wherein each microwell has a hole to the microwell and a hole to the round well-bottom. A cell culture substrate having an intermediate effective diameter between the nadirs (D _half-way ), wherein the ratio of D _top : D _half-way is 1.5 : 2.5. The method of claim 16
D _top is 200 μm to 500 μm, cell culture substrate. The method of claim 16
The cell culture substrate, wherein the height H is 100 μm to 500 μm. The method of claim 16
H = 0.7 to 1.3 D _half-way , cell culture substrate. The method of claim 16
D _half-way is 200 to 1000㎛, cell culture substrate.

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