Apparatus and method for generating and analyzing three-dimensional cellular materials - Patents.com

Abstract

An apparatus is provided for containing three-dimensional cellular material surrounded by a medium, which facilitates and maintains centering of the three-dimensional cellular material throughout an assay. The apparatus includes a well having an open proximal end and a closed distal end. The well further defines a compartment having an inner surface and a sample nesting site for containing the three-dimensional cellular material surrounded by a medium. A central recess is located at the closed distal end of the well, a first concentric lip is located above the central recess in the y-direction toward the open proximal end of the well, and a second concentric lip is located above the first concentric lip in the y-direction toward the open proximal end of the well. Additionally, the first concentric lip and the second concentric lip define a groove therebetween. A method of forming a three-dimensional cellular material is also provided.

Description

[Technical field]

[0001] [Related Applications] This application claims priority to U.S. Provisional Patent Application No. 63 / 276,099, filed November 5, 2021, and U.S. Provisional Patent Application No. 63 / 320,912, filed March 17, 2022, the entireties of which are incorporated herein by reference. [Background technology]

[0002] Although in vitro 3D culture systems are more complex than 2D cell culture systems, they have proven to have several advantages that can mimic in vivo cell behavior. Examples of 3D culture systems include, but are not limited to, spheroids, organoids, and embryoids. Spheroids are 3D cellular materials made from a single cell type, such as an established cell line. Organoids are 3D cellular materials made from several cell types that closely resemble, for example, a target organ. Embryoids are 3D cellular materials made from pluripotent stem cells and contain representative cells of the three germ layers. Cells growing in these scaffold-free platforms generate and organize their own 3D extracellular matrix without added materials, in which cell-cell interactions dominate over cell-substrate interactions, and the 3D structure closely resembles in vivo tissue.

[0003] The advent of ultra-low attachment (ULA) plates has enabled spheroids and other 3D cellular materials to provide a simple means to arrive at 3D cell culture models, thanks to their ease of generation and scalability to high throughput. ULA plates can generate spheroids and other 3D structures that are uniform in terms of size, shape, and time required for spheroid formation. Such consistency has made it possible to employ 3D structures for a variety of applications, such as disease modeling, drug discovery and drug safety, tissue engineering, and regenerative medicine.

[0004] Spheroid microplates for analytical analysis, such as with the Seahorse XFe96 instrument, are now commercially available. Such plates allow for metabolic assessment of single spheroids and microtissues. However, the current plate design has several limitations. First, spheroids cannot be generated or cultured in a generic plate that is subsequently used for metabolic analysis with the Seahorse instrument. Instead, spheroids must first be generated in a culture plate, such as an ultra-low attachment plate or a hanging drop plate, and then manually transferred to another spheroid plate for metabolic analysis. Such manual transfer processes are very tedious, time-consuming, and prone to failure due to loss or damage of the small, fragile spheroids. Furthermore, many of the spheroids that are successfully transferred from the culture plate to the metabolic analysis plate do not remain centered during the mixing cycle that must be completed to perform the metabolic analysis. Such misalignment can lead to unstable metabolic measurements or even a complete loss of signal if the spheroids are completely displaced from the measurement area of ​​the well in the culture plate. Moreover, misalignment can also lead to challenges during imaging of spheroids, as many commercially available plates have a wide base, resulting in spheroids moving out of the focal plane. In addition, current spheroid microplates have a large microchamber volume, which is the temporary volume of liquid that is formed when the sensor cartridge is lowered into the well of the microplate to perform metabolic analysis. This large microchamber volume reduces the sensitivity of metabolic assays and also necessitates the use of larger spheroids with diameters of about 500 micrometers to generate a measurable signal, whereas spheroids or other three-dimensional cellular materials with diameters of about 100 micrometers to about 250 micrometers are preferred. Furthermore, currently available spheroid microplates are not applicable for more sophisticated applications, such as biochip-based metabolic assays or co-culture-based metabolic assays.

[0005] Thus, there is a need for microplates and methods for forming spheroids and other three-dimensional cellular materials that address the challenges discussed above with current microplates for forming, culturing, centering, and assaying three-dimensional cellular materials. Summary of the Invention

[0006] In one embodiment, the present disclosure is directed to an apparatus for containing a three-dimensional cellular material surrounded by a medium. The apparatus includes a well, the well having an open proximal end and a closed distal end defining a bottom of the well. Furthermore, the well defines a compartment having an inner surface and a sample nesting site for containing the three-dimensional cellular material surrounded by a medium. Additionally, a central recess is located at the closed distal end of the well, a first concentric lip is located above the central recess in the y-direction toward the open proximal end of the well, and a second concentric lip is located above the first concentric lip in the y-direction toward the open proximal end of the well. Additionally, the first concentric lip and the second concentric lip define a groove therebetween.

[0007] In one embodiment, the first concentric lip can have a first concave radius of curvature, the second concentric lip can have a second concave radius of curvature, and the central recess can have a third concave radius of curvature.

[0008] In another embodiment, the inner surface of the compartment near the closed distal end of the well can be defined by a first convex radius of curvature between the central recess and the first concentric lip, and a second convex radius of curvature between the first concentric lip and the second concentric lip.

[0009] In yet another embodiment, the bottom of the well may be transparent.

[0010] In yet another embodiment, a coating can be attached to at least a portion of the interior surface of the compartment, and the coating can facilitate collection of the three-dimensional cellular material in a central recess at the closed distal end of the well.

[0011] In yet another embodiment, at least one protrusion may be located at the closed distal end of the well, and the at least one protrusion may be radially spaced from a central recess of the well towards the sidewall.

[0012] In one embodiment, the inner surface may comprise polyethylene terephthalate, polystyrene, polypropylene, polyvinyl chloride, cyclic olefin copolymer, polycarbonate, or combinations thereof.

[0013] In another embodiment, the device can include a probe that forms a seal at the closed distal end of the well when introduced into the compartment, and the first concentric lip or the second concentric lip defines a flat surface for receiving the probe. Additionally, the probe can include a sensor for measuring a parameter, such as a metabolic parameter, and the well can hold a volume of medium below the seal that is less than 200 microliters. For example, the volume can range from about 0.25 microliters to about 1.75 microliters.

[0014] In yet another embodiment, the three-dimensional cellular material may comprise a spheroid, an organoid, or a tissue sample.

[0015] In yet another embodiment, the device may include multiple wells defining multiple compartments.

[0016] In another particular embodiment, the present disclosure is directed to a method for forming a three-dimensional cellular material, the method comprising: a) providing a plate having at least one well, the well having an open proximal end and a closed distal end defining a bottom of the well, the well defining a compartment having an inner surface and a sample nesting site for receiving the three-dimensional cellular material, a central recess located at the closed distal end of the well, a first concentric lip located above the central recess in a y-direction toward the open proximal end of the well, and a second concentric lip located above the first concentric lip in a y-direction toward the open proximal end of the well, the first concentric lip and the second concentric lip defining a groove therebetween; b) adding cells and medium to the compartment; and c) allowing the three-dimensional cellular material to form from the cells.

[0017] In one embodiment, the first concentric lip can have a first concave radius of curvature, the second concentric lip can have a second concave radius of curvature, and the central recess can have a third concave radius of curvature.

[0018] In another embodiment, the inner surface of the compartment near the closed distal end of the well can be defined by a first convex radius of curvature between the central recess and the first concentric lip, and a second convex radius of curvature between the first concentric lip and the second concentric lip.

[0019] In yet another embodiment, the bottom of the well may be transparent.

[0020] In yet another embodiment, a coating can be attached to at least a portion of the interior surface of the compartment, the coating being capable of reducing the level of adhesion of the three-dimensional cellular material to the interior surface.

[0021] In yet another embodiment, at least one protrusion may be located at the closed distal end of the well, and the at least one protrusion may be radially spaced from a central recess of the well towards the sidewall.

[0022] In additional embodiments, the inner surface may include polyethylene terephthalate, polystyrene, polypropylene, polyvinyl chloride, polycarbonate, cyclic olefin copolymer, or combinations thereof.

[0023] In another embodiment, the method can further include measuring a parameter of the three-dimensional cellular material by introducing a probe into the compartment to form a seal near the closed distal end of the well, where the first concentric lip or the second concentric lip defines a flat surface for receiving the probe. Additionally, the probe can include a sensor for measuring a parameter, such as a metabolic parameter. Additionally, the volume of medium contained within the well below the seal can be less than 200 microliters. For example, the volume of medium can range from about 0.25 microliters to about 1.75 microliters.

[0024] In yet another embodiment, the three-dimensional cellular material may comprise a spheroid, an organoid, or a tissue sample.

[0025] In yet another embodiment, the three-dimensional cellular material can have a radius in the x-direction and a radius in the y-direction, where the ratio of the radius in the x-direction to the radius in the y-direction is in the range of about 0.75 to about 1.25 after step (c).

[0026] In yet another embodiment, the plate may include multiple wells defining multiple compartments.

[0027] Other features and aspects of the disclosure are discussed in further detail below.

[0028] A full and enabling disclosure of the present disclosure is set forth in more detail in the remainder of the specification, including references to the accompanying figures. [Brief description of the drawings]

[0029] [Figure 1] FIG. 1 is a top view of one embodiment of a cell culture device as contemplated by the present disclosure. [Diagram 2] 2 is a partial cross-sectional view of the cell culture device shown in FIG. 1 taken along dashed line CC, including an enlarged portion of the cross-sectional view of FIG. 2 showing well geometries contemplated by the present disclosure. [Diagram 3] FIG. 13 is another cross-sectional view of a well of a cell culture device contemplated by the present disclosure. [Figure 4] FIG. 4 is a perspective view of the well of FIG. 3. [Diagram 5] FIG. 4 is a bottom view of the well of FIG. 3. [Figure 6] FIG. 6 is a cross-sectional view of the well of FIG. 5 taken along dashed line 6-6. [Figure 7] FIG. 1 is a top view of one embodiment of a cell culture device contemplated by the present disclosure. [Figure 8] FIG. 1 is an upright, exploded perspective view of a cell culture device (e.g., a multi-well plate) and a cartridge with a cover adapted to mate with the plate, illustrating various features of the cell culture device according to one embodiment of the present disclosure. [Figure 9] FIG. 1 is an exploded, upside-down perspective view of a cell culture device (e.g., a multi-well plate) and a cartridge with a cover adapted to mate with the plate, illustrating various features of the cell culture device according to one embodiment of the present disclosure; [Figure 10] 1 is a schematic diagram of a measurement system and apparatus according to one embodiment of the present disclosure. [Figure 11] Photographs of the top view of spheroids cultured in a device contemplated by the present disclosure versus spheroids cultured in a device contemplated by the present disclosure that were cultured in a different device and then transferred to a well of a device contemplated by the present disclosure. [Figure 12] FIG. 1 is a schematic diagram of an example of a three-dimensional cellular material formed according to methods contemplated by the present disclosure. [Figure 13]1 is a graph showing oxygen consumption rate (OCR) versus time measurements comparing spheroids cultured in different devices and transferred to a device contemplated by the present disclosure with spheroids cultured directly in a device contemplated by the present disclosure. [Figure 14] Photographs of top views of spheroids cultured in a device contemplated by the present disclosure, where each well of the device is smooth and contains 20 microliters of coating material, the left column shows the spheroids before an OCR assay is performed, and the right column shows the spheroids after an OCR assay is performed. [Figure 15] FIG. 15 is a graph showing OCR versus time for the spheroids shown in FIG. [Figure 16] Photographs of the top view of HepG2 spheroids cultured in a device contemplated by the present disclosure, where each well of the device is not smooth and contains 50 microliters of coating material, the left column shows the spheroids before an OCR assay was performed, and the right column shows the spheroids after an OCR assay was performed. [Figure 17] FIG. 17 is a graph showing OCR versus time for the spheroids shown in FIG. [Figure 18] Photographs of the top view of HepG2 spheroids cultured in a device contemplated by the present disclosure, where each well of the device is smooth and contains 50 microliters of coating material, the left column shows the spheroids before an OCR assay is performed, and the right column shows the spheroids after an OCR assay is performed. [Figure 19] 19 is a graph showing OCR versus time for the spheroids shown in FIG. 18. [Figure 20] FIG. 1 is a top view photograph of HepG2 spheroids cultured in a device contemplated by the present disclosure, where the spheroids were generated using centrifugation. [Figure 21]FIG. 13 is a top view photograph of Panc1 spheroids cultured in a device contemplated by the present disclosure, where the spheroids were generated using centrifugation. [Figure 22] Photograph of a top view of C2C12 spheroids cultured in a device contemplated by the present disclosure, where the spheroids were generated using centrifugation. [Figure 23A] FIG. 1 is a top view photograph of HepG2 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with 2-methacryloyloxyethyl phosphorylcholine polymer, i.e., MPC polymer (e.g., Lipidure®). [Figure 23B] FIG. 1 is a top view photograph of HepG2 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with 2-methacryloyloxyethyl phosphorylcholine polymer, i.e., MPC polymer (e.g., Lipidure®). [Figure 23C] FIG. 1 is a top view photograph of HepG2 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with 2-methacryloyloxyethyl phosphorylcholine polymer, i.e., MPC polymer (e.g., Lipidure®). [Figure 24A] FIG. 1 is a top view photograph of HepG2 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®. [Figure 24B] FIG. 1 is a top view photograph of HepG2 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®. [Figure 24C] FIG. 1 is a top view photograph of HepG2 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®. [Diagram 25]1 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from HepG2 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and then centrifuged. [Figure 26] 1 is a graph showing the extracellular acidification rate (ECAR) of spheroids formed from HepG2 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and then centrifuged. [Figure 27] FIG. 27 is a series of top-view photographs of various HepG2 spheroids after completion of the OCR and ECAR assays summarized in FIG. 25 and FIG. 26. [Figure 28A] FIG. 13 is a top view photograph of Panc1 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®, the spheroids having a diameter of approximately 425 micrometers. [Figure 28B] FIG. 13 is a top view photograph of Panc1 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®, the spheroids having a diameter of approximately 550 micrometers. [Figure 28C] FIG. 13 is a top view photograph of Panc1 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®, the spheroids having a diameter of approximately 750 micrometers. [Figure 29] 1 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from Panc1 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of approximately 425 micrometers. [Diagram 30]1 is a graph showing the extracellular acidification rate (ECAR) of spheroids formed from Panc1 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of approximately 425 micrometers. [Diagram 31] 1 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from Panc1 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of approximately 750 micrometers. [Diagram 32] 1 is a graph showing the extracellular acidification rate (ECAR) of spheroids formed from Panc1 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of approximately 750 micrometers. [Figure 33A] FIG. 13 is a top view photograph of HepG2 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®, the spheroids having a diameter of approximately 370 micrometers. [Figure 33B] FIG. 13 is a top view photograph of HepG2 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®, the spheroids having a diameter of approximately 450 micrometers. [Figure 33C] FIG. 13 is a top view photograph of HepG2 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®, the spheroids having a diameter of approximately 525 micrometers. [Diagram 34]1 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from HepG2 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of approximately 370 micrometers. [Diagram 35] 1 is a graph showing the extracellular acidification rate (ECAR) of spheroids formed from HepG2 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of approximately 370 micrometers. [Diagram 36] 1 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from HepG2 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of approximately 525 micrometers. [Figure 37] 1 is a graph showing the extracellular acidification rate (ECAR) of spheroids formed from HepG2 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of approximately 525 micrometers. [Figure 38] FIG. 1 is a series of top-view photographs of various C2C12 spheroids cultured in a device contemplated by the present disclosure, the device being coated with BioFLOAT®, the spheroids having a diameter of approximately 150 micrometers. [Figure 39] FIG. 1 is a top view photograph of uncentrifuged C2C12 spheroids cultured in a device contemplated by the present disclosure, the device being coated with BioFLOAT®. [Diagram 40] FIG. 1 is a top view photograph of uncentrifuged Panc1 spheroids cultured in a device contemplated by the present disclosure, the device being coated with BioFLOAT®. [Diagram 41]FIG. 1 is a bar graph showing that ATP levels are proportional to the size of spheroids; the device used to form the spheroids was coated with BioFLOAT®. [Diagram 42] 1 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from non-centrifuged Panc1 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating, the spheroids having diameters of about 325 micrometers and about 250 micrometers. [Diagram 43] 1 is a graph showing the extracellular acidification rate (ECAR) of spheroids formed from non-centrifuged Panc1 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating, the spheroids having diameters of about 325 micrometers and about 250 micrometers. [Diagram 44] 1 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from non-centrifuged C2C12 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating, the spheroids having diameters of about 180 micrometers and about 140 micrometers. [Diagram 45] 1 is a graph showing the extracellular acidification rate (ECAR) of spheroids formed from non-centrifuged C2C12 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating, the spheroids having diameters of about 180 micrometers and about 140 micrometers. [Figure 46] 46 is an image of a 140 micrometer spheroid that was assayed to determine the OCR and ECAR of FIG. 44 and FIG. 45. [Figure 47] 46 is an image of a 180 micrometer spheroid that was assayed to determine the OCR and ECAR of FIG. 44 and FIG. 45. [Figure 48]44 is an image of a 250 micrometer spheroid that was assayed to determine the OCR and ECAR of FIG. 42 and FIG. 43. [Figure 49] 44 is an image of a 325 micrometer spheroid that was assayed to determine the OCR and ECAR of FIG. 42 and FIG. 43. [Figure 50] 1 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from Panc1 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating, the spheroids having a diameter of approximately 400 micrometers. [Figure 51] FIG. 51 is an image of a 400 micrometer spheroid that was assayed to determine the OCR of FIG. [Figure 52] 1 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from C2C12 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating, the spheroids having a diameter of approximately 180 micrometers. [Diagram 53] FIG. 53 is an image of a 180 micrometer spheroid that was assayed to determine the OCR of FIG. [Figure 54] 1 is a confocal image of spheroids formed from Panc1 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating, the spheroids having a diameter of approximately 350 micrometers. [Figure 55] 1 is a confocal image of spheroids formed from Panc1 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating, the spheroids having a diameter of approximately 500 micrometers. [Figure 56] 1 is a confocal image of spheroids formed from C2C12 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating, the spheroids having a diameter of approximately 100 micrometers. [Figure 57] 1 is a confocal image of spheroids formed from C2C12 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating, the spheroids having a diameter of approximately 150 micrometers. [Figure 58] 1 is a confocal image of spheroids formed from C2C12 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and stained with Cell Tracker Orange. [Figure 59] 1 is a confocal image of spheroids formed from C2C12 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and stained with calcein AM. [Figure 60] 1 is a confocal image of spheroids formed from HepG2 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and stained with Hoechst 34580. [Figure 61] 1 is a graph showing the basal oxygen consumption rate (OCR) of hepatic spheroids obtained and then transferred for culture in a device contemplated by the present disclosure. [Figure 62] FIG. 64 is an image of the spheroid with the lowest OCR from FIG. 63, showing many loose cells at the periphery of the spheroid, indicating that the spheroid may have been damaged during transfer. [Figure 63] FIG. 1 is a graph showing the oxygen consumption rate (OCR) of hepatic spheroids cultured in a device contemplated by the present disclosure, the plate being made from polystyrene and the cartridge for the mitochondria stress test being made from polycarbonate. [Figure 64] FIG. 1 is a graph showing the oxygen consumption rate (OCR) of hepatic spheroids cultured in a device contemplated by the present disclosure, in which the plates and cartridges for the mitochondrial stress test are made from polyethylene terephthalate (PET). [Figure 65]1 is a graph showing the oxygen consumption rate (OCR) of PANC1 spheroids cultured and assayed in a device contemplated by the present disclosure. [Figure 66] 1 is a graph showing the oxygen consumption rate (OCR) during mitochondrial stress testing of non-centrifuged PANC1 spheroids cultured in commercially available plates and then transferred to a device contemplated by the present disclosure for testing. [Figure 67] 1 is a graph showing the oxygen consumption rate (OCR) during mitochondrial stress testing of centrifuged PANC1 spheroids cultured in commercially available plates and then transferred to a device contemplated by the present disclosure for testing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] For simplicity, the data presented can be further refined by implementing geometry-specific algorithmic refinements.

[0031] Repeat use of reference characters in the present specification and drawings is intended to represent same or analogous features or elements of the invention.

[0032] Those skilled in the art should understand that the matters discussed herein are merely descriptions of exemplary embodiments and are not intended to limit the broader aspects of the present disclosure.

[0033] Exemplary aspects of the present disclosure are directed to devices and methods for analyzing one or more metabolic parameters associated with three-dimensional materials (e.g., spheroids, organoids, etc.).The devices and methods of the present disclosure allow for the controlled growth and subsequent metabolic analysis of such three-dimensional materials in a single well plate, without the need for separate plates for growth and analysis, thus eliminating the material transfer step required in many prior systems.

[0034] The device of the present disclosure can be integrated into any suitable system designed to analyze three-dimensional materials.For example, the device of the present disclosure can be integrated into all types of cell metabolism analysis systems, microfluidic systems, microplate readers, multi-mode readers and absorbance readers, and imaging systems.

[0035] One particular device that has made significant advances is the SEAHORSE analysis platform manufactured and sold by Agilent Technologies. The SEAHORSE analysis platform can perform quantitative measurements of, for example, mitochondrial function and cellular bioenergetics. For example, the instrument can measure oxygen concentration and pH in the extracellular medium of cell-based assays. Various aspects of the SEAHORSE analysis platform are described in U.S. Patent No. 7,276,351, U.S. Patent No. 7,638,321, U.S. Patent No. 8,697,431, U.S. Patent No. 9,170,253, U.S. Patent Application Publication No. 2014 / 0170671, U.S. Patent Application Publication No. 2015 / 0343439, U.S. Patent Application Publication No. 2016 / 0077083, and U.S. Patent Application Publication No. 2016 / 0096173, which are incorporated herein by reference in their entirety. The apparatus and methods of the present disclosure can be incorporated into the above-mentioned devices to provide various advantages and benefits.

[0036] The systems and processes of the present disclosure can also be incorporated into microplate readers, including multimode readers and absorbance readers. For example, the detection system of the present disclosure can be incorporated into a variety of exemplary devices, including the SYNERGY Hybrid Multimode Reader, CYTATION Hybrid Multimode Reader, LOGPHASE Microbiology Reader, EPOCH Microplate Spectrophotometers, Elx808 Absorbance Reader, and 800 TS Absorbance Reader, all of which are available through Agilent Technologies.

[0037] In general terms, the present disclosure is directed to an apparatus for containing a three-dimensional cellular material surrounded by a medium. The apparatus includes a well, the well having an open proximal end and a closed distal end defining a bottom of the well. Additionally, the well defines a compartment having an inner surface and a sample nesting site for containing the three-dimensional cellular material surrounded by a medium. Additionally, a central recess is located at the closed distal end of the well, a first concentric lip is located above the central recess in the y-direction toward the open proximal end of the well, and a second concentric lip is located above the first concentric lip in the y-direction toward the open proximal end of the well. Additionally, the first concentric lip and the second concentric lip define a groove therebetween. The present disclosure is also directed to a method for forming a three-dimensional cellular material. The method includes the steps of: a) providing a plate having at least one well, the well having an open proximal end and a closed distal end defining a bottom of the well, the well defining a compartment having an inner surface and a sample nesting site for accommodating three-dimensional cellular material, a central recess located at the closed distal end of the well, a first concentric lip located above the central recess toward the open proximal end of the well in the y-direction, and a second concentric lip located above the first concentric lip toward the open proximal end of the well in the y-direction, the first concentric lip and the second concentric lip defining a groove therebetween; b) adding cells and medium to the compartment; and c) allowing the three-dimensional cellular material to form from the cells.

[0038] Such devices and methods allow the in situ generation of three-dimensional cellular materials such as spheroids and organoids, and the subsequent assay of the three-dimensional cellular materials in the same plate, thus eliminating the incomplete transfer step that may lead to inaccurate assay results. Furthermore, when transfer is required, the transfer is made more efficient and accurate by using a multi-channel pipette or by automation due to a specific geometry of the bottom of the well, which also facilitates the centering of the three-dimensional cellular materials in the cell culture device. Furthermore, the geometry uses gravity to facilitate the in situ generation and automatic centering of the three-dimensional cellular materials during the assay and also during the mixing cycle and imaging. Furthermore, the volume of the microchambers formed during the measurement cycle is about half the volume of a standard commercial well plate, which improves the accuracy of any measurement made due to a high signal / background ratio. This also allows smaller three-dimensional cellular materials to be efficiently analyzed. Additionally, the well plates are specifically designed to allow sensor probes to measure one or more parameters, such as metabolic parameters, and include flat surfaces upon which those probes sit, eliminating the need for complex insertions and the associated cumbersome processes involved in the use of such insertions.

[0039] 1, one embodiment of a cell culture device 100 contemplated by the present disclosure is shown. Although a cell culture device 100 having eight wells 102 is shown, it should be understood that a cell culture device having any number of wells 102 (e.g., 1, 4, 6, 8, 12, 16, 24, 48, 96, 384, 1536, or any other number of wells) is contemplated by the present disclosure. Regardless of the number of wells 102 included in the cell culture device 100, each well 102 can include a central recess 122, a first concentric lip 124, and a second concentric lip 126 positioned outwardly of the first concentric lip 124. Additionally, each well 102 can include one or more peripheral protrusions 144, as shown.

[0040] 2 is a partial cross-sectional view of the cell culture device 100 shown in FIG. 1 taken along dashed line CC, including a partial enlarged view of the cross-sectional view of FIG. 2 showing the geometry of the well 102 contemplated by the present disclosure. The well 102 has an open proximal end 104 in the y-direction and a closed distal end 106, with a sidewall 154 extending therebetween to define an inner surface 118 of the well 102. Additionally, a central recess 122 is located at the bottom 119 of the well 102 at the closed distal end 106 of the well 102, a first concentric lip 124 is located above the central recess 122 toward the open proximal end 104 of the well 102 in the y-direction, and a second concentric lip 126 is located above the first concentric lip 124 toward the open proximal end 104 of the well 102 in the y-direction. Additionally, the first concentric lip 124 and the second concentric lip 126 define a groove 156 therebetween. As shown, the first concentric lip 124 can have a first concave radius of curvature RC1, the second concentric lip 126 can have a second concave radius of curvature RC2, and the central recess 122 can have a third concave radius of curvature RC3. Meanwhile, the inner surface 118 of the well 102 near the closed distal end 106 can be defined by a first convex radius of curvature RC4 located between the central recess 122 and the first concentric lip 124 as well as a second convex radius of curvature RC5 located between the first concentric lip 124 and the second concentric lip 126.

[0041] The first concave radius of curvature RC1 can be about 25 micrometers to about 200 micrometers, for example, about 50 micrometers to about 175 micrometers, for example, about 75 micrometers to about 100 micrometers, or any range therebetween. Furthermore, the second concave radius of curvature RC2 can be about 50 micrometers to about 175 micrometers, for example, about 75 micrometers to about 100 micrometers, or any range therebetween. Furthermore, the third concave radius of curvature RC3 can be about 400 micrometers to about 600 micrometers, for example, about 425 micrometers to about 575 micrometers, for example, about 450 micrometers to about 550 micrometers, or any range therebetween. Furthermore, the first convex radius of curvature RC4 can be about 0.75 millimeters to about 2.25 millimeters, for example, about 1 millimeter to about 2 millimeters, for example, about 1.25 millimeters to about 1.75 millimeters, or any range therebetween. Finally, the second convex radius of curvature RC5 can be about 175 micrometers to about 325 micrometers, for example, about 200 micrometers to about 300 micrometers, for example, about 225 micrometers to about 275 micrometers, or any range therebetween. Without being limited to any particular theory, the inventors have found that such a configuration for the well 102 creates an environment amenable to the formation of three-dimensional cellular materials centered with a more compact geometry that more closely resembles in vivo tissue growth, as compared to the three-dimensional cellular materials formed by currently available cell culture plates that tend to exhibit pancake-like geometries, while also allowing for a reduced medium volume for more accurate assay measurements and improved sensitivity.

[0042] Referring now to FIG. 3, each well 102 can include a coating 120 on the inner surface 118 of the well 102. In particular, the coating 120 can be attached to at least a portion of the inner surface 118. It has been found that the coating 120 can improve the smoothness of the inner surface 118 and can reduce adhesion of any three-dimensional cellular material to the inner surface 118. In some embodiments, the well 102 can be formed from a molded polymer, which can include polyethylene terephthalate, polystyrene, polypropylene, polyvinyl chloride, polycarbonate, cyclic olefin copolymer, or combinations thereof. The coating 120 can be any suitable coating agent, including but not limited to a polymeric coating agent. For example, the polymeric coating agent can be a poloxamer (e.g., Pluronic® F-127 or Pluronic® P-188) or a 2-methacryloyloxyethyl phosphorylcholine polymer, i.e., MPC polymer (e.g., Lipidure® from AMSBIO). Other examples of suitable coating agents include RinseAid from STEMCELL Technologies and BioFLOAT® from faCellitate. Additionally, it should be appreciated that the bottom 119 of the well 102 can be transparent to allow for imaging or other photometric measurements. Additionally, in some embodiments, the interior surface 118 of the well 102 can be smooth to enhance the transparency of the well 102.

[0043] 4, a perspective view of the well 102 of FIG. 3 is shown, and FIG. 5 is a bottom view of the well 102 of FIG. 3. As shown, the well 102 includes a compartment 140, with the central recess 122, the first concentric lip 124, and the second concentric lip 126 detailed in the perspective view, the first concentric lip 124 and the second concentric lip 126 defining a groove 156 therebetween. Additionally, as shown in FIG. 5, the well 102 can include one or more peripheral projections 144 located at the closed distal end 106 of the well 106, which can be radially spaced from the central recess 122 of the well 102 toward a sidewall 154 (see FIG. 6). The inventors have found that the central recess 122 can confine movement of the spheroid or other three-dimensional cellular material 300 (see FIG. 6) within the well 102 to a limited space or area, e.g., corresponding to the focal plane of any imaging device, such that the spheroid or other three-dimensional cellular material 300 remains centered, while the protrusion 144 can prevent damage to any existing coating, such as an ultra-low adhesion coating, by the test cartridge.

[0044] FIG. 6 is a cross-sectional view of the well 102 of FIG. 5 along dashed line 6-6, in which the three-dimensional cellular material 300 is contained within a sample nesting site 112 formed within the compartment 140 by a central recess 122 and surrounded by a medium 148 (e.g., cell culture medium). Furthermore, when the probe 114 is inserted into the well 102, the space defined by the probe 114 and the central recess 122 creates a microchamber 146 to contain the three-dimensional cellular material 300 within the medium 148 for analysis of a parameter. Based on the geometric configuration of the well 102 of the present disclosure, the inventors have discovered that the volume of the microchamber 146 can be significantly reduced to provide more accurate parameter measurements. For example, the volume of the microchamber 146 can be less than 200 microliters, e.g., less than 100 microliters, e.g., less than 50 microliters, e.g., less than 25 microliters, e.g., less than 10 microliters, e.g., less than 5 microliters, e.g., less than 2 microliters. For example, the volume can be from about 0.25 microliters to about 1.75 microliters, e.g., from about 0.3 microliters to about 1.5 microliters, e.g., from about 0.4 microliters to about 1.4 microliters, or any range therebetween. While any parameter can be measured by the method described in more detail below with respect to Figures 8-10, in some embodiments, the parameters are metabolic parameters such as oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and pH. As shown, the probe 114 can form a seal 152 at the closed distal end 106 of the well 102 when introduced into the compartment 140, and the first concentric lip 124 or the second concentric lip 126 can create a flat surface 150 for receiving the probe 114, depending on the size of the probe 114 utilized.

[0045] As shown in Figure 7, it should be understood that the present disclosure contemplates a cell culture device 200 that can be in the form of a well plate similar to device 100 shown in Figure 1, but includes wells 202 interconnected by channels 204 between adjacent wells. Such a configuration allows for the use of live-on-a-chip applications in conjunction with the methods contemplated by the present disclosure, allowing for simultaneous metabolic analysis of multi-organoid-based live-on-a-chip co-culture models.

[0046] In any event, referring to Figures 11 and 12, compared to the more pancake-like and non-uniform geometric shapes of materials cultured in other cell culture devices (the two left sets of images in Figure 11), the three-dimensional cellular material 300 formed by the device 100, 200, or any other device contemplated by the present disclosure (the one right set of images in Figure 11) can be compact and of uniform size and shape in terms of the radius of the three-dimensional cellular material 300 in the x-direction Rx and the y-direction Rc. For example, the ratio of the radius Rx in the x-direction to the radius Ry in the y-direction can be about 0.75 to about 1.25, such as about 0.8 to about 1.2, such as about 0.85 to about 1.15, such as about 0.9 to about 1.1, or any range therebetween. Further, the three-dimensional cellular material 300 can have a radius (or diameter) Rx or Ry from about 25 micrometers to about 500 micrometers, such as from about 30 micrometers to about 400 micrometers, such as from about 40 micrometers to about 300 micrometers, such as from about 50 micrometers to about 250 micrometers, or any range therebetween.

[0047] 8 and 9 are upright and upside-down exploded perspective views, respectively, of a cell culture device (e.g., a multi-well plate) and a cartridge with a cover adapted to mate with the plate, illustrating various features of the cell culture device according to one embodiment of the present disclosure.

[0048] 8 and 9, a well plate configuration suitable for performing the above-mentioned parametric tests and implementing the embodiments of the present disclosure is shown. The well plate includes a cell culture device 100 that defines a plurality of wells 102. The cell culture device 100 can be combined with a cartridge 128 and a removable cover 142. In the illustrated embodiment, the cell culture device 100 has 24 wells, but it should be understood that the number of wells 102 in the plate can vary from one to several thousand. In some embodiments, a single well of almost any size can be made, or several wells can be made, or several wells can be made in a one- or two-dimensional arrangement. In various embodiments, a two-dimensional pattern of wells can be developed that corresponds to the pattern and dimensions of a microplate, for example, as described in the Society for Biomolecular Screening's microplate specifications ("SBS-1 Footprints" and "SBS-4 Well Positions", both of which are detailed draft specifications updated on May 20, 2003). The plate may include 1, 4, 6, 8, 12, 16, 24, 48, 96, 384, 1536, or any other number of individual wells. A larger number of wells poses engineering challenges due to the fine structures required to implement the embodiments of the present disclosure. The cartridge 128 is a generally flat element with a frame 130, for example made from molded plastic. The flat surface 132 defines a number of regions 134 that correspond to or are aligned with some of the respective openings of the multiple wells 102 defined in the cell culture device 100. In the illustrated embodiment, in each of those regions 134, the flat element defines a first, second, third, and fourth port 136 that serves as a reservoir for the delivery of gas or reagents, and a central opening 138 for a probe 114 that houses one or more sensors 116. Each of the ports is adapted to hold and release test fluid onto a respective well 102 below on demand.The ports 136 are sized and positioned such that a group of four ports can be positioned above the wells 102 and a gas or test fluid can be delivered to each well 102 from any one of the four ports. In some embodiments, the number of ports in each region can be less than or greater than four. The ports 136 and probes 114 can be flexibly mounted to the cell culture device 100 to accommodate lateral movement and thereby accommodate the microplate. By constructing the microplate to include a flexible region, its manufacturing tolerances can be relaxed, allowing the cartridge to be used with microplates of slightly different dimensions. Flexibility can be achieved by forming flat surfaces 132, for example with an elastomeric polymer, in each region to allow relative movement between the frame 130 and the probes 114 and ports 136.

[0049] Each of the ports 136 may have a cylindrical, conical, or cubic shape, open at the top through the flat surface 132, and closed at the bottom except for a small hole, i.e., a capillary opening, typically centered within the bottom surface. The capillary opening is adapted to hold the test fluid within the port by surface tension in the absence of an external force, e.g., a positive pressure differential force, a negative pressure differential force, or possibly a centrifugal force. Each port may be made from a polymeric material that is impermeable to gas, test compounds, or any other solid material. When configured for use with the cell culture device 100, the volume of liquid accommodated by each port may range from less than 200 microliters, e.g., less than 100 microliters, e.g., less than 50 microliters, e.g., less than 25 microliters, e.g., less than 10 microliters, e.g., less than 5 microliters, e.g., less than 2 microliters. For example, the volume can be from about 0.25 microliters to about 1.75 microliters, such as from about 0.3 microliters to about 1.5 microliters, such as from about 0.4 microliters to about 1.4 microliters, or any range therebetween.

[0050] FIG. 10 shows an overview of a measurement system and device (e.g., analyzer) for use with embodiments of the present disclosure. It comprises an analyzer 160 including a compound storage and delivery device 162 disposed in a housing 164 (shown in dashed lines), a cartridge 128 defining a plurality of openings and a plurality of flexibly mounted fluid ports (shown in detail in FIGS. 8 and 9) for receiving a sensor structure, and a stage or base 166 adapted to receive a cell culture device 100, e.g., a cell culture plate. The cartridge 128 is disposed above the cell culture device 100 and adapted to mate therewith. The cartridge 128 is optionally held by a cartridge holder 168 adapted to receive the cartridge 128. The device also includes a mounting block 170, which is preferably motor (not shown) driven to reciprocate as indicated by the double-headed arrow, and includes a lifting mechanism 172. The lifting mechanism 172 may be adapted to move the cartridge 128 relative to the stage 166 or the cell culture device 100. The mounting block 170 includes a gas multiplexer 174 attached to a gas source or gas reservoir 176. The gas source 176 is in fluid communication with the cartridge 128 and is used to drive the delivery of test fluids from ports of the cartridge 128 to the wells 102 of the cell culture device 100 or to immobilize gas compositions in one or more wells 102. A plurality of probes 114 having sensors 116 are adapted to be inserted into a plurality of openings in the cartridge 128 and can be used to collect data indicative of the status of cells disposed in the wells of the cell culture device 100.

[0051] The compound storage and delivery device 162 is controlled by a controller 178, which may be integral to a computer 180, which may control the lifting mechanism 172, the multiplexer 174, and the gas source or reservoir 176. In doing so, the controller 178 may enable delivery of test fluid from a port 136 to a corresponding well 102 when an associated sensor 116 is positioned in the well 102.

[0052] The device described herein is a modification of the device disclosed in Published Patent Application No. 2008 / 0014571, which allows for experimentation and analysis of 3D cell culture samples, such as tissue samples, biopsy samples, or cell scaffolds holding cells, the disclosure of which is incorporated herein by reference. The viability of the sample can be maintained and control can be exercised over its microenvironment. In some embodiments, the microenvironment around the sample can be modified by adding gas to the medium or to the headspace of the well above the medium to alter the composition of dissolved gases. In some other embodiments, the microenvironment around the sample can be modified by adding a solution of a biologically active substance to the medium to expose the sample to the biologically active substance. A metered amount of one or more gases and / or one or more drugs or other solutes can be added to the medium of the well to set the microenvironment of the medium around the sample to a predetermined point. The microenvironment of the well can be set to a hypoxic state. The concentration of one or more solutes in the medium around the sample can be measured. The concentration of the solutes in the medium around the sample can be measured multiple times at time intervals.

[0053] A commonly performed test for the systems described above is the mitochondrial stress test. In that assay, a series of injections are delivered through the drug ports of the cartridge to measure the response of a biological sample to various compounds (oligomycin, FCCP, rotenone, and antimycin). The compounds are preloaded into the drug reservoirs (ports) on the XF cartridge prior to running the assay. When the cartridge is inserted into the instrument, it is coupled to a manifold that, when activated by a solenoid valve, provides air pressure to the headspace of the reservoir to push the compounds through small orifices into the wells that contain the biological samples. The air manifold and valve system can be modified to redirect one of their ports to an external gas source (gas cylinder or bottle). The gas source can be connected to the instrument through a port on the rear connector panel. The bottle can be located near the instrument and can include a regulator and bubbler for humidification of the incoming gas. The solenoid valve can be activated to open, allowing gas to flow through the manifold / cartridge interface, through the drug port orifice, and into the headspace of the biological sample. By rocking the probe vertically, the gas mixes with the medium, allowing control of the oxygen available to the sample. For example, by flowing argon into the headspace, the available oxygen in the medium is displaced, creating a more hypoxic condition around the sample. Ambient levels of oxygen can be restored by stopping the gas and mixing.

[0054] In some embodiments, a solution source of a biologically active substance may be in fluid communication with the medium of the well for exposing the sample to the substance.

[0055] To control the operation and timing of the solenoid valves, the software of the instrument can be modified to facilitate valve / timing control and to indicate some of the calculation variables used during calibration. For example, to calculate the molar concentration of oxygen in the medium, the concentration at the time of calibration is preferably known and entered into a calculation table. Under some conditions, the initial calibration value (F or current ambient concentration) may not be known. In that case, calibration can be achieved by injecting sodium sulfite into a set of control wells and calibrating the system based on the known values. As will be understood by those skilled in the art, certain coefficients can be made accessible in the software to calculate those results. Separate windows can be created in the software to facilitate access to those variables, valve control, and calculation of the calibration coefficients.

[0056] The instrument can be tested with a well-characterized cell line to verify proper operation and control of the gas system. A series of tests can be performed to demonstrate the ability to purge oxygen from the medium and create a hypoxic microenvironment around the sample. These tests can include 1. calibration of the instrument under known and unknown ambient O2 concentrations and 2. verification of the performance of the gas delivery system and its ability to bring environmental oxygen levels to the desired value (<5% PPO). This can be verified within the instrument by referencing oxygen level data. Readings from the instrument can provide a chart representing that data.

[0057] An alternative to controlling oxygen and pH in the sample environment would be to enclose the entire instrument in an environmental chamber and evacuate the chamber to the desired level. Such an alternative approach may be undesirable as it can be very costly, occupy a lot of laboratory space, and take a long time to achieve the desired levels around the tissue. By the time such oxygen levels are achieved, the tissue may die. EXAMPLES

[0058] The following examples illustrate certain illustrative preferred embodiments and applications of the present disclosure, but do not represent all embodiments and applications.

[0059] [Example 1] First, Panc1 / HepG2 cells were grown in a well plate generally contemplated by the present disclosure but having only a single concentric lip as well as in a commercially available plate, and then the spheroids cultured in the commercially available plate were transferred to the plate of the present disclosure. The experimental protocol was as follows. 1. Add 50ul of Lipidure solution (prepared in ETOH) to each plate. Leave at 37°C overnight or 50°C for 1 hour to evaporate the ETOH. 2. Trypsinize HepG2 and wash several times with Mg-Ca-free PBS to make single cells. Place in an incubator at 37°C for 2-3 minutes. 3. Transfer the cells into a centrifuge tube, add PBS to fill the tube halfway, then count the cells based on density before centrifugation. 4. Centrifuge the cell suspension and resuspend the pellet in fresh medium. 5. Add 10K cells per well along with fresh growth medium (180ul per well). 6. If necessary, centrifuge at reduced speed at 1000 rpm for 2-3 minutes. Centrifugation may be required to help clump the cells together. 7. When desirable dense masses have formed (3-5 days), proceed to the Seahorse XF assay. 8. For HepG2, DMEM medium by Seahorse, pH 7.4, supplemented with glutamine, sodium pyruvate, and glucose was used (same concentrations as growth medium). 9. Using several channels, remove approximately 150 microliters of spent medium and add 180 microliters of fresh Seahorse medium by gently sliding it down the walls. 10. Next, wash 2-3 times to dilute the FBS in the wells. 11. After each wash, check to see if the spheroids are still centered. The white dots of the spheroids are easily visible on the dark lab countertop. 12. Follow known protocols for conventional oxygen consumption rate (OCR) assays based on the mitochondrial stress test detailed above. 13. Take before and after images to verify that the spheroid remains centered throughout. 14.DMEM contains high glucose for Panc1 cells as opposed to low glucose for HepG2.

[0060] [Results and Observations] As can be seen in Figure 11, the spheroids generated in the Agilent plates were very similar to those generated in the Corning and Insphero plates, but with improved alignment and greater uniformity in terms of geometry. The difference in spheroid size reflects the difference in geometry of the different ULA plates.

[0061] 13, an oxygen consumption rate assay was performed using the mitochondrial stress test described above using Panc1 spheroids generated in Corning plates and plates having the geometric configuration of the present disclosure. The difference in oxygen consumption rate is due to spheroids in the plates of the present disclosure having a diameter of about 150 micrometers to about 200 micrometers compared to spheroids grown in Corning plates having a diameter of about 200 micrometers to about 250 micrometers, which has a more pancake-like geometry as shown in FIG.

[0062] 14 and 15, using Panc1 spheroids generated in plates with the geometric configuration of the present disclosure and assayed after 8 days, oxygen consumption rates were performed on wells 1-6 using the mitochondrial stress test described above. The spheroids showed good responses to FCCP and antimycin A / rotenone, which is also typically seen with 2D Panc1 cells. Note that the laboratory Panc1 cells were oligomycin resistant and therefore did not show a decrease in oxygen consumption. The plates had a smooth surface with 20 microliters of Lipidure coating.

[0063] Further, with reference to Figures 16 and 17, oxygen consumption rates were performed on wells 1-6 using the mitochondrial stress test described above using HepG2 spheroids generated in plates with the geometric configuration of the present disclosure and assayed after 6 days. The spheroids showed good responses to oligomycin, FCCP, and antimycin A / rotenone, which is also typically seen with 2D HepG2 cells. The plates had a non-smooth surface with 50 microliters of Lipidure coating. In Figure 16, the spheroids in wells 1, 3, 4, and 5 appear to have moved from the center after the assay. However, as can be seen in Figure 17, this can be easily interpreted as a displacement occurring while the sensor cartridge was removed from the plate after the assay.

[0064] Finally, with reference to Figures 18 and 19, oxygen consumption rates were performed on wells 1-6 using the mitochondrial stress test described above using HepG2 spheroids generated in plates with the geometric configuration of the present disclosure and assayed after 6 days. The spheroids showed good responses to oligomycin, FCCP, and antimycin A / rotenone, which is also typically seen with 2D HepG2 cells. The plates have a smooth surface with 50 microliters of Lipidure coating.

[0065] [Example 2] Panc1, HepG2, and C2C12 cell lines were used to form spheroids in devices having wells with at least the geometries shown in Figures 2, 3, and 4 of this disclosure. The wells were coated with Lipidure® or BioFLOAT® ultra-low adhesion coating according to the manufacturer's protocol, after which the cell lines were introduced into the well plates and spheroids were formed according to the following protocol. · ULA coating is applied according to manufacturer's protocol Use trypsin to detach the cells of interest (C2C12, Panc1, or HepG2) and create a single cell suspension. Inactivate trypsin by adding complete culture medium. -Perform cell counting. · Centrifuge the cell suspension at 250G for 5 minutes and resuspend the pellet in fresh medium at the required density. Add 300-1500 cells per well with fresh growth medium to a final volume of 200ul per well. If necessary, centrifuge at 250 g for 5 minutes at low acceleration. Centrifugation may be required to help disaggregate the cells. When the desired dense mass has formed (typically 3-5 days), proceed to the Seahorse XF assay. · DMEM medium by Seahorse, pH 7.4, supplemented with glutamine, sodium pyruvate, and glucose was used (same concentrations as growth medium). Using several channels, remove approximately 100 microliters of spent medium and add 100 microliters of fresh Seahorse medium by gently sliding it down the walls. · Next, wash 3 to 5 times to dilute the old medium in the wells. · Perform the desired Seahorse assay - basal, mitochondria stress test, FCCP addition, or other.

[0066] The resulting spheroids remained in the center of the well during assays using the SEAHORSE analysis platform, allowing detection of basal metabolic signals. Furthermore, spheroids of various sizes could be generated, with one spheroid per well being possible without centrifugation, as evidenced by the figures and graphs discussed below.

[0067] In particular, Figures 20, 21 and 22 show successful generation of spheroids in wells of a plate contemplated by the present disclosure using three different cell lines. For example, Figure 20 is a top view of a HepG2 spheroid cultured in a device contemplated by the present disclosure, where the spheroids were generated using centrifugation. Meanwhile, Figure 21 is a top view of a Panc1 spheroid cultured in a device contemplated by the present disclosure, where the spheroids were generated using centrifugation. Additionally, Figure 22 is a top view of a C2C12 spheroid cultured in a device contemplated by the present disclosure, where the spheroids were generated using centrifugation.

[0068] Next, Figures 23A, 23B, and 23C show HepG2 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with 2-methacryloyloxyethyl phosphorylcholine polymer, i.e., MPC polymer (e.g., Lipidure®).

[0069] Furthermore, Figure 24A, Figure 24B, and Figure 24C show HepG2 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®. Figure 25 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from HepG2 cells cultured in a device contemplated by the present disclosure with BioFLOAT® coating and then centrifuged, and Figure 26 is a graph showing the extracellular acidification rate (ECAR) of spheroids formed from HepG2 cells cultured in a device contemplated by the present disclosure with BioFLOAT® coating and then centrifuged. These graphs show that when spheroids are cultured in the wells of a device contemplated by the present disclosure, a signal of basal metabolism can be measured. Additionally, FIG. 27 is a series of top-view photographs of various HepG2 spheroids after the OCR and ECAR assays summarized in FIG. 25 and FIG. 26 were completed, which demonstrate that the spheroids remained centered during the metabolic assays.

[0070] In addition, Figures 28A, 28B, and 28C demonstrate that spheroids of various sizes can be generated, and Figures 29, 30, 31, and 32 demonstrate that basal metabolic signals can be measured for Panc1 spheroids cultured in a device contemplated by the present disclosure. For example, Figure 28A shows a Panc1 spheroid cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®, the spheroid having a diameter of about 425 micrometers. Furthermore, Figure 28B shows a Panc1 spheroid cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®, the spheroid having a diameter of about 550 micrometers. Next, Figure 28C shows Panc1 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®, the spheroids having a diameter of about 750 micrometers.Furthermore, Figure 29 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from Panc1 cells cultured in a device contemplated by the present disclosure with a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of about 425 micrometers, and Figure 30 is a graph showing the extracellular acidification rate (ECAR) of spheroids formed from Panc1 cells cultured in a device contemplated by the present disclosure with a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of about 425 micrometers.Additionally, FIG. 31 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from Panc1 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of approximately 750 micrometers, and FIG. 32 is a graph showing the extracellular acidification rate (ECAR) of spheroids formed from Panc1 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of approximately 750 micrometers.

[0071] In addition, Figure 33A, Figure 335B, and Figure 33C demonstrate that spheroids of various sizes can be generated, and Figure 34, Figure 35, Figure 36, and Figure 37 demonstrate that basal metabolic signals can be measured for HepG2 spheroids cultured in a device contemplated by the present disclosure. For example, Figure 33A shows a HepG2 spheroid cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®, the spheroid having a diameter of about 370 micrometers. Furthermore, Figure 33B shows a HepG2 spheroid cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®, the spheroid having a diameter of about 450 micrometers. Next, Figure 33C shows HepG2 spheroids cultured in a device contemplated by the present disclosure and then centrifuged, the device being coated with BioFLOAT®, the spheroids having a diameter of about 525 micrometers. Further, Figure 34 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from HepG2 cells cultured in a device contemplated by the present disclosure with a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of about 370 micrometers, and Figure 35 is a graph showing the extracellular acidification rate (ECAR) of spheroids formed from HepG2 cells cultured in a device contemplated by the present disclosure with a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of about 370 micrometers.Additionally, FIG. 36 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from HepG2 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of approximately 525 micrometers, and FIG. 37 is a graph showing the extracellular acidification rate (ECAR) of spheroids formed from HepG2 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and then centrifuged, the spheroids having a diameter of approximately 525 micrometers.

[0072] This example also demonstrates that smaller spheroids can be formed in the wells of a device contemplated by the present disclosure. For example, Figure 38 shows a series of top-view photographs of various C2C12 spheroids cultured in a device contemplated by the present disclosure, the device being coated with BioFLOAT®, with the spheroids having a diameter of approximately 150 micrometers.

[0073] This example further demonstrates that the wells of a device of the present disclosure facilitate the formation of a single spheroid per well, even without centrifugation. For example, Figure 39 is a top-view photograph of a non-centrifuged C2C12 spheroid cultured in a device contemplated by the present disclosure, which device has been coated with BioFLOAT®, and Figure 40 is a top-view photograph of a non-centrifuged Panc1 spheroid cultured in a device contemplated by the present disclosure, which device has been coated with BioFLOAT®.

[0074] [Example 3] First, C2C12 and Panc1 cells were plated at 600 cells / well (for smaller spheroids) or 1200 cells / well (for larger spheroids) on BioFLOAT® coated spheroid plates as contemplated by the present disclosure. No centrifugation was used and cells were allowed to form spheroids for 3 days. On the third day, ATP levels were measured using Promega's Cell Titer Glo 3D kit for spheroids of various sizes. Figure 41 is a bar graph showing that ATP levels are proportional to the size of the spheroids. Specifically, the larger the spheroid, the higher the luminescence value and therefore the higher the ATP levels for both C2C12 and Panc1 cell types.

[0075] Next, C2C12 cells and Panc1 cells were seeded at two different concentrations on spheroid plates coated with BioFLOAT® as contemplated by the present disclosure. The cells were allowed to settle without centrifugation and form spheroids for two days. Then, OCR and ECAR assays were performed, including both basal measurements and FCCP injection (final concentration 1 μM). FCCP, an uncoupler that collapses the proton gradient and destroys the mitochondrial membrane potential to release the inhibition of the electron transport chain, was used to trigger maximum respiration in cells. To characterize the size of the spheroids, the maximum diameter of the cross section was measured. Panc1 spheroids are about 325 micrometers in diameter for larger spheroids and about 250 micrometers in diameter for smaller spheroids, while the large C2C12 spheroids are about 180 micrometers in diameter and the small C2C12 spheroids are about 140 micrometers in diameter. FIG. 42 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from Panc1 cells, the spheroids had diameters of about 325 micrometers and about 250 micrometers. FIG. 43 is a graph showing the extracellular acidification rate (ECAR) of spheroids formed from Panc1 cells, the spheroids had diameters of about 325 micrometers and about 250 micrometers. FIG. 44 is a graph showing the oxygen consumption rate (OCR) of spheroids formed from C2C12 cells, the spheroids had diameters of about 180 micrometers and about 140 micrometers. FIG. 45 is a graph showing the extracellular acidification rate (ECAR) of spheroids formed from C2C12 cells, the spheroids had diameters of about 180 micrometers and about 140 micrometers. As shown, larger spheroids for both cell types had higher OCR and ECAR compared to smaller spheroids for both cell types, and both OCR and ECAR were elevated after FCCP injection.

[0076] After the OCR and ECAR assays were completed, the spheroids were imaged using bright field imaging on a BioTek Cytaion1 imager, as shown in Figures 46-49. Figure 46 is an image of a 140 micrometer spheroid that was assayed to determine the OCR and ECAR of Figures 44 and 45. Figure 47 is an image of a 180 micrometer spheroid that was assayed to determine the OCR and ECAR of Figures 44 and 45. Figure 48 is an image of a 250 micrometer spheroid that was assayed to determine the OCR and ECAR of Figures 42 and 43. Figure 49 is an image of a 325 micrometer spheroid that was assayed to determine the OCR and ECAR of Figures 42 and 43.

[0077] Successful growth and measurement of spheroids in the same plate was then demonstrated. Panc1 spheroids were grown in BioFLOAT® coated spheroid plates as contemplated by the present disclosure until they reached a size of approximately 400 micrometers in diameter. On the day of the assay, the medium was replaced with Seahorse DMEM. The plates were moved to a 37°C incubator without CO2 for 1 hour. Mitostress tests were performed, including injections of oligomycin, FCCP, and rotenone / antimycin. Figure 50 is a graph showing each injection point and the subsequent oxygen consumption rate (OCR) of spheroids formed from Panc1 cells cultured in a device contemplated by the present disclosure. As shown, additional measurement cycles were added to allow time for the drugs to diffuse into the spheroids. The spheroids responded well to all drugs and remained in place throughout the measurement. Figure 51 is an image of a 400 micrometer spheroid assayed to determine the OCR shown in Figure 50.

[0078] Successful growth and measurement of spheroids in the same plate was then demonstrated. As contemplated by the present disclosure, C2C12 spheroids were grown in BioFLOAT® coated spheroid plates until they reached a size of approximately 180 micrometers in diameter. On the day of the assay, the medium was replaced with Seahorse DMEM. The plates were moved to a 37°C incubator without CO2 for 1 hour. Mitostress tests were performed, including injections of oligomycin, FCCP, and rotenone / antimycin. Figure 52 is a graph showing each injection point and the subsequent oxygen consumption rate (OCR) of spheroids formed from C2C12 cells cultured in a device contemplated by the present disclosure. As shown, additional measurement cycles were added to allow time for the drugs to diffuse into the spheroids. The spheroids responded well to all drugs and remained in place throughout the measurements. FIG. 53 is an image of the 180 micrometer spheroids that were assayed to determine the OCR shown in FIG.

[0079] Further, spheroids of two different cell types were grown at various cell numbers in BioFLOAT® coated spheroid plates as contemplated by the present disclosure. The live spheroids were then stained with CyQuant Direct Cell Proliferation Dye and imaged by confocal on a BioTek Cytation 10. As shown in Figures 54-57, good staining indicates that the cells are viable, and also confirms the suitability of the spheroids and plates as contemplated by the present disclosure for the confocal imaging process. In particular, Figure 54 is a confocal image of a spheroid formed from an initial seeding of 600 Panc1 cells cultured in a device as contemplated by the present disclosure with a BioFLOAT® coating, and the spheroid had a diameter of about 350 micrometers. Figure 55 is a confocal image of a spheroid formed from an initial seeding of 1200 Panc1 cells cultured in a device contemplated by the present disclosure with a BioFLOAT® coating, the spheroid had a diameter of about 500 micrometers. Figure 56 is a confocal image of a spheroid formed from an initial seeding of 600 C2C12 cells cultured in a device contemplated by the present disclosure with a BioFLOAT® coating, the spheroid had a diameter of about 100 micrometers. Figure 57 is a confocal image of a spheroid formed from an initial seeding of 1200 C2C12 cells cultured in a device contemplated by the present disclosure with a BioFLOAT® coating, the spheroid had a diameter of about 150 micrometers.

[0080] Further, additional fluorophores were used to characterize live C2C12 spheroids grown in spheroid plates coated with BioFLOAT® coating as contemplated by the present disclosure. Confocal imaging of both Cell Tracker Orange and Calcein AM allowed clear visualization of live spheroids by Z-projection images as shown. In particular, FIG. 58 is a confocal image of spheroids formed from C2C12 cells cultured in a device contemplated by the present disclosure with BioFLOAT® coating and stained with Cell Tracker Orange, and FIG. 59 is a confocal image of spheroids formed from C2C12 cells cultured in a device contemplated by the present disclosure with BioFLOAT® coating and stained with Calcein AM.

[0081] Finally, FIG. 60 is a confocal image of spheroids formed from HepG2 cells cultured in a device contemplated by the present disclosure having a BioFLOAT® coating and stained with Hoechst 34580, showing a z-projection image of stitched tiled images, clearly demonstrating the ability to visualize individual cell nuclei of living spheroids.

[0082] [Example 4] Spheroids can also be generated in other spheroid plates and transferred to the spheroid plates described herein for measurement. Such a workflow has the added challenge of ensuring that the spheroids are not damaged and lost during the transfer process. Such protocols are challenging for very small spheroids that are difficult to see by eye, as it is necessary to visualize the spheroids being transferred at the tip of the pipette. A centrifugation step after transfer is also required to generate useful data. Exemplary transfer protocol: Fill the wells of the plate described in this invention with 170 ul of Seahorse medium. Pipette 20ul of medium / spheroid from the plate containing pre-grown spheroids. Make sure the spheroid is at the tip of the pipette. Allow the spheroids to settle to the tip by gravity. Dispense the minimum amount of medium that will allow the spheroids to be transferred to new wells. For better quality data, centrifuge at 200g for 5 minutes with soft brakes. Incubate for 1 hour at 37°C in a CO2-free incubator. · Perform Seahorse assay.

[0083] To assess transfer function, liver spheroids (300 μm) were purchased from InSphero and maintained according to the manufacturer's protocol until the time of assay. As shown in Figure 61, the basal OCR response was not consistent across all spheroids. Specifically, some spheroids had lower signals than others (lines closest to the x-axis). As shown in Figure 62, spheroids were imaged after the Seahorse assay to assess morphology, and it was observed that there were many loose cells around the outside of the spheroids, suggesting that they may have been damaged during the transfer process.

[0084] [Example 5] The plates described in this invention can be made from a variety of materials, with the most suitable material depending on the final measurement application. Plates were used to measure spheroids molded in both polystyrene and polyethylene terephthalate, grown by InSphero, and transferred to the plate according to the protocol in Example 4. As shown in Figures 63 and 64, both polystyrene and polyethylene terephthalate plates allow good OCR measurements of spheroids, but the measured signal is 1.5 times higher for the polyethylene terephthalate plate (Figure 64) compared to the polystyrene plate (Figure 63).

[0085] [Example 6] To demonstrate the advantage of growing and measuring spheroids in the same plate, we performed a side-by-side comparison of spheroids grown directly in the plates described herein versus spheroids transferred from a commercial spheroid plate. In both cases, the spheroids were grown following the same protocol with initial seeding of Panc1 cells. Figures 65-67 show the results of the Seahorse assay, including a comparison of the OCR signal for PANC1 spheroids grown in the plates disclosed herein versus spheroids grown in a commercial plate and transferred to the plates disclosed herein with and without centrifugation for measurement in the Seahorse mitostress test. Figure 65 shows the results when spheroids are grown on the plates. Figure 66 shows the results when spheroids are transferred to the plates and centrifuged prior to assay. Figure 67 shows the results when spheroids are transferred and not centrifuged prior to assay. Well-to-well variability is reduced when a centrifugation step is added. Spheroids grown in this plate showed more consistent results with no damaged spheroids observed.

[0086] Throughout, applicants have demonstrated a suitable plate configuration for generating spheroids that remain centrally collected within the well, thereby allowing metabolic measurements of the spheroids to be made. The plate design is also suitable for viewing the centrally collected spheroids with an imaging device.

[0087] These and other modifications and variations to the present disclosure, as more particularly set forth in the appended claims, may be implemented by those skilled in the art without departing from the spirit and scope of the present disclosure. It is further to be understood that aspects of the various embodiments may be interchanged in whole or in part. Moreover, those skilled in the art will appreciate that the above description is merely illustrative and is not intended to limit the present disclosure, as more particularly set forth in the appended claims.

Claims

1. 1. A device for containing three-dimensional cellular material surrounded by culture medium, the device comprising a well having an open proximal end and a closed distal end defining a bottom of the well, the well defining a compartment having an inner surface and a sample nesting site for containing the three-dimensional cellular material surrounded by culture medium, a central recess located at the closed distal end of the well, a first concentric lip located above the central recess toward the open proximal end of the well in a y-direction, and a second concentric lip located above the first concentric lip toward the open proximal end of the well in the y-direction, the first concentric lip and the second concentric lip defining a groove therebetween.

2. 2. The apparatus of claim 1, wherein the first concentric lip has a first concave radius of curvature, the second concentric lip has a second concave radius of curvature, and the central recess has a third concave radius of curvature.

3. 2. The device of claim 1, wherein the interior surface of the compartment near the closed distal end of the well is defined by a first convex radius of curvature between the central recess and the first concentric lip, and a second convex radius of curvature between the first concentric lip and the second concentric lip.

4. The device of claim 1 , wherein the bottom of the well is transparent.

5. The device of claim 1 , wherein a coating is attached to at least a portion of the interior surface of the compartment, the coating facilitating collection of the three-dimensional cellular material in the central recess at the closed distal end of the well.

6. 2. The device of claim 1, wherein at least one protrusion is located at the closed distal end of the well, the at least one protrusion being radially spaced from the central recess toward a sidewall of the well.

7. The device of claim 1 , wherein the inner surface comprises polyethylene terephthalate, polystyrene, polypropylene, polyvinyl chloride, cyclic olefin copolymer, polycarbonate, or a combination thereof.

8. 2. The device of claim 1, further comprising a probe that forms a seal at the closed distal end of the well when introduced into the compartment, wherein the first concentric lip or the second concentric lip defines a flat surface for receiving the probe.

9. The apparatus of claim 8 , wherein the probe comprises a sensor for measuring a parameter.

10. 9. The device of claim 8, wherein the well holds a volume of medium below the seal that is less than 200 microliters.

11. 11. The device of claim 10, wherein the volume of the medium below the seal ranges from about 0.25 microliters to about 1.75 microliters.

12. The device of claim 1 , wherein the three-dimensional cellular material comprises a spheroid, an organoid, or a tissue sample.

13. The device of claim 1 , wherein the device comprises a plurality of wells defining a plurality of compartments.

14. 1. A method for forming a three-dimensional cellular material, the method comprising: a) providing a plate having at least one well, the well having an open proximal end and a closed distal end defining a bottom of the well, the well defining a compartment having an inner surface and a sample nesting site for accommodating the three-dimensional cellular material, a central recess located at the closed distal end of the well, a first concentric lip located above the central recess toward the open proximal end of the well in a y-direction, and a second concentric lip located above the first concentric lip toward the open proximal end of the well in the y-direction, the first concentric lip and the second concentric lip defining a groove therebetween; b) adding cells and medium to said compartment; c) allowing the three-dimensional cellular material to form from the cells; A method comprising:

15. 15. The method of claim 14, wherein the first concentric lip has a first concave radius of curvature, the second concentric lip has a second concave radius of curvature, and the central recess has a third concave radius of curvature.

16. 15. The method of claim 14, wherein the interior surface of the compartment near the closed distal end of the well is defined by a first convex radius of curvature between the central recess and the first concentric lip, and a second convex radius of curvature between the first concentric lip and the second concentric lip.

17. The method of claim 14, wherein the bottom of the well is transparent.

18. 15. The method of claim 14, wherein a coating is attached to at least a portion of the interior surface of the compartment, the coating reducing the level of adhesion of the three-dimensional material and / or other cellular material to the interior surface.

19. 15. The method of claim 14, wherein at least one protrusion is located at the closed distal end of the well, the at least one protrusion being radially spaced from the central recess toward a sidewall of the well.

20. 15. The method of claim 14, wherein the interior surface comprises polyethylene terephthalate, polystyrene, polypropylene, polyvinyl chloride, polycarbonate, cyclic olefin copolymer, or a combination thereof.

21. 15. The method of claim 14, further comprising measuring a parameter of the three-dimensional cellular material by introducing a probe into the compartment to form a seal near the closed distal end of the well, wherein the first concentric lip or the second concentric lip defines a flat surface for receiving the probe.

22. 22. The method of claim 21, wherein the probe comprises a sensor for measuring the parameter.

23. 22. The method of claim 21, wherein the volume of medium contained in the well below the seal is less than 200 microliters.

24. 24. The method of claim 23, wherein the volume of the medium ranges from about 0.25 microliters to about 1.75 microliters.

25. 15. The method of claim 14, wherein the three-dimensional cellular material comprises a spheroid, an organoid, or a tissue sample.

26. 15. The method of claim 14, wherein the three-dimensional cellular material has a radius in an x ​​direction and a radius in a y direction, and the ratio of the radius in the x direction to the radius in the y direction is in the range of about 0.75 to about 1.25 after step (c).

27. The method of claim 14 , wherein the plate comprises a plurality of wells defining a plurality of compartments.

28. 28. An analytical system utilizing the device of any one of claims 1 to 13 or the method of any one of claims 14 to 27.

29. 30. The analytical system of claim 28, wherein the analytical system comprises a cellular metabolism analytical system, a microfluidic system, a microplate reader, a multimode reader, an absorbance reader, an imaging system, or a combination thereof.

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