Patent ID: 12203059

DETAILED DESCRIPTION

Reference will now be made in greater detail to various embodiments of the subject matter of the present disclosure, some embodiments of which are illustrated in the accompanying drawings. Like numbers used in the figures refer to like components, steps and the like. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. In addition, the use of different numbers to refer to components is not intended to indicate that the different numbered components cannot be the same or similar to other numbered components.

The present disclosure describes, among other things, cell culture apparatuses having a structured bottom surface defining a shape of a plurality of wells or microwells. In some embodiments, a substrate forming the wells can comprise an exterior surface that defines an external surface of the apparatus. The shape of the external surface can be controlled to facilitate imaging of cells within the wells in accordance with various embodiments described herein.

In some embodiments, the wells may be configured such that cells cultured in the wells form spheroids. For example, the wells may be non-adherent to cells to cause the cells in the wells to associate with each other and form spheres. The spheroids may expand to size limits imposed by the geometry of the cells. In some embodiments, the wells may be coated with an ultra-low binding material to make the wells non-adherent to cells.

In some embodiments, the inner surface of the wells may be non-adherent to cells. The wells may be formed from non-adherent material or may be coated with non-adherent material to form a non-adherent well. In some embodiments, the non-adherent material may be described as an ultra-low-adhesion material. Examples of non-adherent material include perfluorinated polymers, olefins, or like polymers or mixtures thereof. Other examples may include agarose, non-ionic hydrogels such as polyacrylamides, or like materials or mixtures thereof. The combination of, for example, non-adherent wells, well geometry, and gravity may induce cells cultured in the wells to self-assembly into spheroids.

However, well geometries that can be useful for culturing spheroids can be difficult to image, either manually or via automated processes, with conventional microscopy techniques due to light distortions introduced through lens like effects by each individual well.

The well or well array design described herein may make image analysis of in vitro 3-dimensional spheroid based assays or spheroid production possible or more feasible. The cross-sectional profile of an individual well may have an impact on quality of imaging capabilities, e.g., microscope imaging capabilities. Specifically, controlling the well thickness and outer shape of the well may help compensate for light path deviation during imaging to improve image quality and may make the cell culture system amenable to high content imaging screening. As a result, the well thickness and outer shape of the well may lead to a well that is optically active due to the lens shape. In other words, the well may be able to utilize one or a variety of light sources and still produce uniform illumination of the cells in the well. In some embodiments, an improved illumination may allow for a shorter focal length, which may increase the NA of the system and allow image acquisition at higher magnifications.

A variety of well characteristics may have a significant impact on imaging quality. For example, dimensions and shape of an interior surface of the well, dimensions and shape of an exterior surface of the well, optical properties of the material defining the well, the thickness profile of the material defining the well, etc. can all play a role in high quality microscopy imaging. Additionally, the refractive index of a material may have a significant impact on imaging quality in both reflective and transmittance microscopy applications. For example, in the case of many cell culture imaging applications, the most common material that is in contact with the interior surface of the well is a water-based solution with a refractive index of 1.33 and the most common material for the well fabrication is polystyrene with a refractive index of 1.59. The differences in refractive indexes of the two materials may cause any incident light beam to deflect/reflect and may result in a negative impact on the microscope image quality.

One way to improve the quality of cell culture images may be to correct the light distortion. The light distortion may be corrected by controlling and varying the well characteristics discussed above. Specifically, the dimensions and shape of the interior and exterior surfaces of the well and the thickness profile of the material defining the well. Previously published fabrication methods have focused on the dimensions and shape of the interior surface of the well, especially, the interior surface that defines the dimensions of the 3D cellular aggregates. However, adjusting any of these characteristics in relation to one another may help to compensate for any light distortion that may occur during imaging (e.g., microscopy, etc.). More specifically, and as described herein, the light distortion may be corrected by controlling the shape and dimensions of the exterior surface of the well. In other words, the ability to change the shape and dimensions of the exterior surface may be utilized to help control the angle at which incident light exits the exterior surface.

A cell culture apparatus100including a plurality of wells115is shown inFIG.1. The plurality of wells115may be defined by a substrate110, e.g., a polymeric material. Each well115may define an interior surface120, an exterior surface114, an upper aperture118, a nadir116, and an upper edge121. The substrate110may define a thickness111between the interior surface120and the exterior surface114. The wells115may have a depth d defined by a height from the nadir116to the upper aperture118. The wells115may also have a diametric dimension w, such as a diameter, width, etc., across the well115defined by the upper aperture118.

In some embodiments, the wells115described herein may define a diametric dimension w of about, e.g., greater than or equal to 100 micrometers, greater than or equal to 300 micrometers, greater than or equal to 500 micrometers, greater than or equal to 800 micrometers, greater than or equal to 1200 micrometers, etc. or, less than or equal to 3000 micrometers, less than or equal to 2600 micrometers, less than or equal to 2200 micrometers, less than or equal to 1800 micrometers, less than or equal to 1500 micrometers, etc., including ranges between any of the foregoing values. Such diametric dimensions can control the size of a spheroid grown therein such that cells at the interior of the spheroid are maintained in a healthy state. In some embodiments, the wells115may define a depth d, by way of example, greater than or equal to 100 micrometers, greater than or equal to 300 micrometers, greater than or equal to 500 micrometers, greater than or equal to 800 micrometers, greater than or equal to 1200 micrometers, etc. or, less than or equal to 3000 micrometers, less than or equal to 2600 micrometers, less than or equal to 2200 micrometers, less than or equal to 1800 micrometers, less than or equal to 1500 micrometers, etc., including ranges between any of the foregoing values. Of course, other suitable dimensions may also be employed.

The exterior surface of the well may be a variety of shapes. For example, the shape of the exterior surface may be configured to correct for refraction of light passing into the interior surface of the well and out of the exterior surface of the well or vice versa. In other words, the light passing out of the exterior surface of the well is substantially parallel to the light passing into the interior surface and/or the shape of the exterior surface may be configured to minimize refraction of light that passes between the interior and exterior surfaces and/or the exterior surface may be configured to transmit light substantially parallel to a direction that the light was received by the interior surface of the well. In some embodiments, the well contains a cell culture medium, and the shape of the exterior surface corrects for refraction.

The thickness of the substrate between the interior surface of the well and the exterior surface of the well may vary. For example, the thickness of the substrate may be configured to correct for refraction of light passing into the interior surface of the well and out of the exterior surface of the well or vice versa. In other words, the light passing out of the exterior surface of the well is substantially parallel to the light passing into the interior surface or the thickness of the substrate may be configured to minimize refraction of light that passes there between. In some embodiments, the well contains a cell culture medium when the thickness of the substrate corrects for refraction.

The cross-sections of two wells200that define an exterior surface214that does not correct for refraction are shown inFIGS.2A and2B. In other words, light that enters201the interior surface220of the well200is not parallel with light that exits202the exterior surface214of the well200.

As shown inFIG.2A, the thickness of the substrate210proximate the nadir216is less than the thickness of the substrate210proximate the upper edge221of the well200. The thickness of the substrate210proximate the upper edge221of the well200may be defined as a thickness between the interior surface220and the exterior surface214on a same plane as the upper aperture218. As shown inFIG.2B, the exterior surface214of the well200defines a rectangular shaped bottom of the substrate210that creates a flat exterior surface of the well200.

In some embodiments, the thickness and shape of the substrate, e.g., a polymeric material, that defines the well may be configured to correct for refraction of light passing into the interior surface of the well and out of the exterior surface of the well. The cross-sections of two embodiments of wells115that define an exterior surface114that does correct for refraction are shown inFIGS.3A and3B. In other words, light that enters201the interior surface120of the well115is parallel with light that exits202the exterior surface114of the well115. In yet other words, a shape of the interior surface120of the well115and a shape of the exterior surface114of the well115are configured to minimize the effects of the refraction of light that passes there between.

As shown inFIGS.3A and3B, the thickness111of the substrate110proximate the nadir116may be greater than or equal to the thickness109of the substrate110proximate the upper aperture118. The thickness of the substrate110proximate the nadir116may be defined as a distance between the interior surface120and the exterior surface114at a lowest point of the well115. The thickness of the substrate110proximate the upper aperture118may be defined as a thickness between the interior surface120and the exterior surface114on a same plane as the upper aperture118.

Specifically, as shown inFIG.3A, the thickness of the substrate110remains constant from proximate the upper aperture118to the nadir116and, as shown inFIG.3B, the thickness111of the substrate110proximate the nadir116is greater than the thickness109of the substrate110proximate the upper aperture118. Also, as shown inFIG.3A, the thickness111of the substrate110proximate to the nadir116may be equal to the thickness109of the substrate110proximate the upper aperture118. The substrate thicknesses shown inFIGS.3A and3Ballow for an incoming light201entering the interior surface120to be substantially parallel to an outgoing light202leaving the exterior surface114.

In other embodiments, the substrate thickness may be described as increasing continuously from proximate the upper aperture to the nadir (e.g.,FIG.3B). The thickness of the substrate proximate any location from the upper aperture to the nadir may be defined by a thickness of, e.g., greater than or equal to 5 micrometers, greater than or equal to 10 micrometers, greater than or equal to 20 micrometers, greater than or equal to 40 micrometers, greater than or equal to 60 micrometers, etc. or, less than or equal to 100 micrometers, less than or equal to 90 micrometers, less than or equal to 80 micrometers, less than or equal to 65 micrometers, less than or equal to 50 micrometers, etc., including ranges between any of the foregoing values. In some embodiments, the thickness is about 1000 micrometers or less. In some embodiments, the thickness is in a range from 10 micrometers to 1000 micrometers.

In some embodiments, the well may define an axis105between the nadir and a center of the upper aperture and the well may be rotationally symmetrical about the axis105(see, e.g.,FIG.1). For example, a hemispherical shape may define the well. The hemispherical shape may be defined by a radius of about, e.g., greater than or equal to 50 micrometers, greater than or equal to 150 micrometers, greater than or equal to 250 micrometers, greater than or equal to 400 micrometers, greater than or equal to 600 micrometers, etc. or, less than or equal to 1500 micrometers, less than or equal to 1300 micrometers, less than or equal to 1100 micrometers, less than or equal to 900 micrometers, less than or equal to 750 micrometers, etc.

Orthogonal views of 3D datasets of X-ray computed tomography images of wells of generally as depicted inFIG.3Aare shown inFIGS.4A-4D. The images depict wells115defining a convex exterior surface114as described inFIG.3A.FIG.4Adepicts a cross sectional view of three complete wells115along horizontal line117, shown inFIG.4C.FIG.4Cis a top view of a portion of a cell culture apparatus with an array of wells115.FIG.4Bis a cross sectional view of wells115along vertical line119shown inFIG.4C.FIG.4Dis a reconstituted 3D image of a portion of a cell culture apparatus with an array of wells115.

Orthogonal views of 3D datasets of X-ray computed tomography images of wells of generally as depicted inFIG.2Bare shown inFIGS.5A-5D. The images depict wells115defining a flat exterior surface114as described inFIG.2B.FIG.5Adepicts a cross sectional view of wells115along horizontal line117shown inFIG.5C.FIG.5Cof a portion of a cell culture apparatus with an array of wells115.FIG.5Bis a cross sectional view of wells115along vertical line119shown inFIG.5C.FIG.5Dis a reconstituted 3D image of a portion of a cell culture apparatus with an array of wells115.

Bright field microscopy images of wells115having shapes generally in accordance withFIGS.3A and2Bare shown inFIGS.6A and6B, respectively. The microscopy images ofFIG.6Ashows light that passed through wells having a shape as depicted inFIG.3A. The microscopy images ofFIG.6Bshows light that passed through wells having a shape as depicted inFIG.2B. The shape of the well as depicted inFIG.3Adid not substantially reflect/deflect and thus yielded a relatively uniform signal across all wells as compared to the signal across wells having a shape as depicted inFIG.2B. More light was received by the microscope camera for wells having a shape as depicted inFIG.3A(seeFIG.6A) than the wells having a shape as depicted inFIG.2Bas shown in microscopy images ofFIG.6B. In other words, the well microscopy images ofFIG.6Bdepicts that more light was scattered, as shown by the dark rings, than the well microscopy images ofFIG.6A.

As shown inFIG.7, the cell culture apparatus700may include a reservoir725.

The reservoir may include a bottom705and an enclosing sidewall720. The bottom705may be defined by a plurality of wells715. Each well715may have similar characteristics as wells described herein (see, e.g.,FIGS.1,3A, and3B).

In some embodiments, the exterior surface of the well is optimized through ray tracing for diffraction limited imaging performance when viewed under high resolution microscopy (e.g., bright field, fluorescence, confocal, or other microscopy modalities). For example, with reference toFIG.2AandFIG.2B, the exterior surface214is optimized through ray tracing.

To illustrate this approach, an interior surface of a polystyrene well may be a hemisphere with a radius of 500 micrometers and a center thickness of 150 micrometers. The diameter of a spheroid may be 300 micrometers, and a 20× microscope with an objective numerical aperture of 0.4 is employed. There may be a number of image points across positions of the spheroid, for example, center, 50 micrometers from the center, 100 micrometers from the center, and 150 micrometers from the center. In such instances, most images taken will be sub-optimum. Spot diagrams can be generated from the different field positions and compared to the diffraction limited Airy circle at the image plane to assess image quality. If the exterior surface is flat, as illustrated inFIG.2B, the spot diameters across the field are a few times larger than the diffraction limit, indicating poor image quality. When the well has a uniform thickness, the image quality is considerably better than the previous case. However, diffraction limited imaging performance is barely achieved within the center 50 micrometer radius. Outside this field of view, astigmatism deteriorates the image quality. However, by optimizing the radius of curvature of the exterior surface to 0.518 mm, the image quality can achieve diffraction limited performance across the entire spheroid diameter, although a small amount of distortion and astigmatism still exist. To further optimize the image quality, an aspheric exterior surface is used. With a radius of curvature R=0.682 mm and a conic constant of K=−3.09, the residual aberration and distortion throughout the entire field of interest are removed. The conic surface is given by:
y2−2Rx+(K+1)x2=0.

Diffraction limited performance is also maintained in the entire volume of the spheroid. This enables high resolution confocal imaging in any locations within the spheroid. The actual magnification is 21.5× due to the refractive effect of the surface.

In some embodiments, nested wells are employed, whereby a first well or layer of wells is present above a second well or layer of wells. Well sidewalls of each well are selected such that light passing through two or more layers of wells remains substantially parallel to the original light.

Any suitable process can be used to fabricate cell culture apparatuses having wells as described herein. For example, a substrate can be molded to form the well or structured surface, a substrate film can be embossed to form the well or structured surface, or the like. In some embodiments, a deforming process is used to fabricate wells as described herein.

For example and with reference toFIG.8, a schematic side view of a deforming process for fabrication of wells is shown. For example,FIG.8illustrates a hot embossing and film deforming process800for the fabrication of thin wall wells. The process uses a thin film820and applies heat and pressure810down onto the thin film820into the mold830. The thin film820may have a specific thickness that results in a given thickness attributed to different sections of the wells. For example, a 70 micrometer thin film after going through a process of hot embossing and film deforming may have a uniform thickness of 25 micrometers at a bottom part of the well and upper part of the well. This outcome is similar to the well shown inFIG.3A, which can sufficiently correct for light refraction. As a result, the hot embossing and film deforming process may be actively controlled during well fabrication to form wells that correct light refraction sufficiently similar to those inFIGS.3A-3B. The well fabrication process may also be performed in planar configuration or as a roll to roll process.

Cell culture apparatuses having wells or structured surfaces as described herein can be formed from any suitable material. Preferably, materials intended to contact cells or culture media are compatible with the cells and the media. Typically, cell culture components (e.g., wells) are formed from polymeric material. Examples of suitable polymeric materials include polystyrene, polymethylmethacrylate, polyvinyl chloride, polycarbonate, polysulfone, polystyrene copolymers, fluoropolymers, polyesters, polyamides, polystyrene butadiene copolymers, fully hydrogenated styrenic polymers, polycarbonate PDMS copolymers, and polyolefins such as polyethylene, polypropylene, polymethyl pentene, polypropylene copolymers and cyclic olefin copolymers, and the like.

Cells cultured in three dimensions, such as spheroids, can exhibit more in vivo like functionality than their counterparts cultured in two dimensions as monolayers. In two dimensional cell culture systems, cells can attach to a substrate on which they are cultured. However, when cells are grown in three dimensions, such as spheroids, the cells interact with each other rather than attaching to the substrate. Cells cultured in three dimensions more closely resemble in vivo tissue in terms of cellular communication and the development of extracellular matrices. Spheroids thus provide a superior model for cell migration, differentiation, survival, and growth and therefore provide better systems for research, diagnostics, and drug efficacy, pharmacology, and toxicity testing.

In some embodiments, the devices are configured such that cells cultured in the devices form spheroids. For example, the wells in which cells are grown can be non-adherent to cells to cause the cells in the wells to associate with each other and form spheres. The spheroids expand to size limits imposed by the geometry of the wells. In some embodiments, the wells are coated with an ultra-low binding material to make the wells non-adherent to cells.

Examples of non-adherent material include perfluorinated polymers, olefins, or like polymers or mixtures thereof. Other examples include agarose, non-ionic hydrogels such as polyacrylamides, polyethers such as polyethylene oxide and polyols such as polyvinyl alcohol, or like materials or mixtures thereof. The combination of, for example, non-adherent wells, well geometry (e.g., size and shape), and/or gravity induce cells cultured in the wells to self-assemble into spheroids. Some spheroids maintain differentiated cell function indicative of a more in vivo-like, response relative to cells grown in a monolayer. Other cells types, such as mesenchymal stromal cells, when cultured as spheroids retain their pluripotency.

In some embodiments, the systems, devices, and methods herein comprise one or more cells. In some embodiments, the cells are cryopreserved. In some embodiments, the cells are in three dimensional culture. In some such embodiments, the systems, devices, and methods comprise one or more spheroids. In some embodiments, one or more of the cells are actively dividing. In some embodiments, the systems, devices, and methods comprise culture media (e.g., comprising nutrients (e.g., proteins, peptides, amino acids), energy (e.g., carbohydrates), essential metals and minerals (e.g., calcium, magnesium, iron, phosphates, sulphates), buffering agents (e.g., phosphates, acetates), indicators for pH change (e.g., phenol red, bromo-cresol purple), selective agents (e.g., chemicals, antimicrobial agents), etc.). In some embodiments, one or more test compounds (e.g., drug) are included in the systems, devices, and methods.

A wide variety of cell types may be cultured. In some embodiments, a spheroid contains a single cell type. In some embodiments, a spheroid contains more than one cell type. In some embodiments, where more than one spheroid is grown, each spheroid is of the same type, while in other embodiments, two or more different types of spheroids are grown. Cells grown in spheroids may be natural cells or altered cells (e.g., cell comprising one or more non-natural 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., embryonic stem cell, induced pluripotent stem cell) in any desired state of differentiation (e.g., pluripotent, multi-potent, fate determined, immortalized, etc.). 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 into a hyper-proliferative state (e.g., transformed cells). Cells may be from or derived from any desired tissue or organ type, including but not limited to, adrenal, bladder, blood vessel, bone, bone marrow, brain, cartilage, cervical, corneal, endometrial, esophageal, gastrointestinal, immune system (e.g., T lymphocytes, B lymphocytes, leukocytes, macrophages, and dendritic cells), liver, lung, lymphatic, muscle (e.g., cardiac muscle), neural, ovarian, pancreatic (e.g., islet cells), pituitary, prostate, renal, salivary, skin, tendon, testicular, and thyroid. In some embodiments, the cells are mammalian cells (e.g., human, mice, rat, rabbit, dog, cat, cow, pig, chicken, goat, horse, etc.).

The cultured cells find use in a wide variety of research, diagnostic, drug screening and testing, therapeutic, and industrial applications.

In some embodiments, the cells are used for production of proteins or viruses. Systems, devices, and methods that culture 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 viruses for vaccine production may be grown in the cells and isolated or purified for use as desired. In some embodiments, the protein is a native protein to the cells. In some embodiments, the protein is non-native. In some embodiments, the protein is expressed recombinantly. Preferably, the protein is overexpressed using a non-native promoter. The protein may be expressed as a fusion protein. 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 separation of the fusion partners after purification.

In some embodiments, the protein is a therapeutic protein. Such proteins include, but are not limited to, proteins and peptides that replace a protein that is deficient or abnormal (e.g., insulin), augment an existing pathway (e.g., inhibitors or agonists), provide a novel function or activity, interfere with a molecule or organism, or deliver other compounds or proteins (e.g., radionuclides, cytotoxic drugs, effector proteins, etc.). In some embodiments, the protein is an immunoglobulin such as an antibody (e.g., monoclonal antibody) of any type (e.g., humanized, bi-specific, multi-specific, etc.). Therapeutic protein categories include, but are not limited to, antibody-based drugs, Fc fusion proteins, anticoagulants, antigens, blood factor, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. Therapeutic proteins may be used to prevent or treat cancers, immune disorders, metabolic disorders, inherited 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 (e.g., receptor-binding ligands), inhibitors, antagonists, and the like. In some embodiments, the diagnostic protein is expressed with or is a detectable moiety (e.g., fluorescent moiety, luminescent moiety (e.g., luciferase), colorimetric moiety, etc.).

In some embodiments, the protein is an industrial protein. Industrial proteins include, but are not limited to, food components, industrial enzymes, agricultural proteins, analytical enzymes, etc.

In some embodiments, the cells are used for drug discovery, characterization, efficacy testing, and toxicity testing. Such testing includes, but is not limited to, pharmacological effect assessment, carcinogenicity assessment, medical imaging agent characteristic assessment, half-life assessment, radiation safety assessment, genotoxicity testing, immunotoxicity testing, reproductive and developmental testing, drug interaction assessment, dose assessment, adsorption assessment, disposition assessment, metabolism assessment, elimination studies, etc. Specific cells types may be employed for specific tests (e.g., hepatocytes for liver toxicity, renal proximal tubule epithelial cells for nephrotoxicity, vascular endothelial cells for vascular toxicity, neuronal and glial cells for neurotoxicity, cardiomyocytes for cardiotoxicity, skeletal myocytes for rhabdomyolysis, etc.). Treated cells may be assessed for any number of desired parameters including, but not limited to, membrane integrity, cellular metabolite content, mitochondrial functions, lysosomal functions, apoptosis, genetic alterations, gene expression differences, and the like.

In some embodiments, the cell culture devices are a component of a larger system. In some embodiments, the system comprises a plurality (e.g., 2, 3, 4, 5, . . . , 10, . . . , 20, . . . , 50, . . . , 100, . . . , 1000, etc.) of such cell culture devices. In some embodiments, the system comprises an incubator for maintaining the culture devices at optimal culture conditions (e.g., temperature, atmosphere, humidity, etc.). In some embodiments, the system comprises detectors for imaging or otherwise analyzing cells. Such detectors include, but are not limited to, fluorimeters, luminometers, cameras, microscopes, plate readers (e.g., PERKIN ELMER ENVISION plate reader; PERKIN ELMER VIEWLUX plate reader), cell analyzers (e.g., GE IN Cell Analyzer 2000 and 2200; THERMO/CELLOMICS CELLNSIGHT High Content Screening Platform), and confocal imaging systems (e.g., PERKIN ELMER OPERAPHENIX high throughput content screening system; GE INCELL 6000 Cell Imaging System). In some embodiments, the system comprises perfusion systems or other components for supplying, re-supplying, and circulating culture media or other components to cultured cells. In some embodiments, the system comprises robotic components (e.g., pipettes, arms, plate movers, etc.) for automating the handing, use, and/or analysis of culture devices.

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

As used herein, singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a “structured bottom surface” includes examples having two or more such “structured bottom surfaces” unless the context clearly indicates otherwise.

As used in this specification and the appended claims, the term “or” is generally employed 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 any two or more of the listed elements.

As used herein, “have”, “has”, “having”, “include”, “includes”, “including”, “comprise”, “comprises”, “comprising” or the like are used in their open ended inclusive sense, and generally mean “include, but not limited to”, “includes, but not limited to”, or “including, but not limited to”.

“Optional” or “optionally” means that the subsequently described event, circumstance, or component, can or cannot occur, and that the description includes instances where the event, circumstance, or component, occurs and instances where it does not.

The words “preferred” and “preferably” refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the inventive technology.

Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, examples include from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Where a range of values is “greater than”, “less than”, etc. a particular value, that value is included within the range.

Any direction referred to herein, such as “top,” “bottom,” “left,” “right,” “upper,” “lower,” “above,” below,” and other directions and orientations are described herein for clarity in reference to the figures and are not to be limiting of an actual device or system or use of the device or system. Many of the devices, articles or systems described herein may be used in a number of directions and orientations. Directional descriptors used herein with regard to cell culture apparatuses often refer to directions when the apparatus is oriented for purposes of culturing cells in the apparatus.

Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that any particular order be inferred. Any recited single or multiple feature or aspect in any one claim can be combined or permuted with any other recited feature or aspect in any other claim or claims.

It is also noted that recitations herein refer to a component being “configured” or “adapted to” function in a particular way. In this respect, such a component is “configured” or “adapted to” embody a particular property, or function in a particular manner, where such recitations are structural recitations as opposed to recitations of intended use. More specifically, the references herein to the manner in which a component is “configured” or “adapted to” denotes an existing physical condition of the component and, as such, is to be taken as a definite recitation of the structural characteristics of the component.

While various features, elements or steps of particular embodiments may be disclosed using the transitional phrase “comprising,” it is to be understood that alternative embodiments, including those that may be described using the transitional phrases “consisting” or “consisting essentially of,” are implied. Thus, for example, implied alternative embodiments to a cell culture apparatus comprising a structured bottom surface, one or more sidewalls, a top and a port include embodiments where a cell culture apparatus consists of a structured bottom surface, one or more sidewalls, a top and a port and embodiments where a cell culture apparatus consists essentially of a structured bottom surface, one or more sidewalls, a top and a port.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present inventive technology without departing from the spirit and scope of the disclosure. Since modifications, combinations, sub-combinations and variations of the disclosed embodiments incorporating the spirit and substance of the inventive technology may occur to persons skilled in the art, the inventive technology should be construed to include everything within the scope of the appended claims and their equivalents.