Apparatus for metabolic analysis of biological samples

Abstract

Apparatus for metabolic analysis of a biological sample such as a three-dimensional biological sample have a microplate comprising a plurality of wells and rings with screens attached. Each of the walls of the wells includes a reservoir wall section adjacent to a bottom end, a shoulder section adjacent to the reservoir wall section, and the ring is positioned on the shoulder section. Analysis systems comprise the apparatus and a device for metabolic measurement. Methods of using the apparatus and analysis system are also provided.

Description

Attorney Docket No. : AGT-21-PCT (20250068-02) APPARATUS FOR METABOLIC ANALYSTS OF BIOLOGICAL SAMPLESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of and priority to US Provisional Patent Application No. 63 / 790,561 having a filing date of April 17, 2025, which is incorporated herein in its entirety by reference thereto.FIELD OF THE INVENTION

[0002] The present disclosure relates to apparatus for metabolic analysis of biological samples.BACKGROUND OF THE INVENTION

[0003] Microplates are widely used for obtaining measurements of cells and other samples, and they are commercially available in a variety of well formats. Microplates for cell cultures are available in 6-, 8-, 12-, 24-, 48-, 96-, 384- and 1536-well formats, and coated and non-coated plates are available for adherent cell cultures and suspension cultures.

[0004] Microplates are often used for conducting measurements on cell populations.Typically, cells are seeded into a well of a microplate by pipetting an aqueous medium comprising cells into the well, and the cells settle on the bottom surface of the well. The seeded microplate is often placed into an incubator to promote cellular growth and expansion. This results in cells covering some or all of the bottom surface of the well. However, there are challenges that arise when trying to obtain high-quality images of cells seeded on the bottom surface of a well.20250068-01 / 027644-8511

[0005] The Seahorse XF96 “Extracellular Flux Analyzer” was launched in 2008 by Seahorse Biosciences. Since that time the analyzer has been adopted for making quantitative measurements of mitochondrial function and cellular bioenergetics. The Seahorse XF analyzer can measure the concentrations of various analytes (O2, H+, CO2) in the extracellular medium of a cell based assay. Analyte concentrations can be measured non-invasively, within a small volume about the cells, providing quantitative measurements for changes in analyte concentrations as a function of time from which bioenergetic flux (example: dO2 / dt=oxygen consumption rate, dpH / dt=extracellular acidification rate) can be determined. The XF measurements generally are based on a method in which a small, temporary, measurement volume is created around the cells, or a sensor is placed in close proximity to the cells.Measurements under these conditions amplifies changes in concentrations, allowing highly sensitive, time resolved measurements to be collected from a set of optical sensors. Once measurement(s) is made, the plunger is lifted and the medium around the cells is restored to its original condition. This nondestructive method allows multiple measurements to be serially collected for a biological sample under various conditions.

[0006] By making metabolic measurements such as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), a profile of the bioenergetic phenotype may be developed based on the substrate and pathway (glycolysis or oxidative phosphorylation) for generating energy and biosynthesis. The analyzer allows quantitative measurements of mitochondrial function and cellular bio-energetics of cells.

[0007] However, there remains a need in the art for apparatus and methods for obtaining metabolic measurements of biological samples, especially three-dimensional biological samples, including aerobic metabolism, glycolysis and intermediary metabolism.20250068-01 / 027644-8511SUMMARY OF THE INVENTION

[0008] In one aspect, the present disclosure provides a method and system for measuring oxygen consumption (OCR) and acidification (ECAR) with higher sensitivity, allowing the researcher to differentiate between different compounds, doses, sizes and treatments. The design includes a PES capture screen with pore openings of 285uM to allow for thorough perfusion of the sample chamber, including media and oxygen turnover and full mixing of injected compounds into the sample chamber to allow for measurement of 3D and tissue samples that require such thorough perfusion. The distance between the sample and the XF sensor is selected to provide higher sensitivity, allowing for robust measurements of OCR and ECAR.

[0009] These and other features and advantages of the present apparatus and methods will be apparent from the following detailed description, in conjunction with the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. l is a view of an embodiment of the present microplate.

[0011] FIG. 2 shows an embodiment of a well in the present microplate.

[0012] FIG. 3 shows an embodiment of a ring comprising a screen of the present apparatus.

[0013] FIG. 4 illustrates an embodiment of the present apparatus with a ring comprising a screen placed in a well.

[0014] FIG. 5 illustrates an embodiment of the present apparatus with a plunger in a well for metabolic measurements.

[0015] FIGS. 6 to 25 show results obtained use of the present apparatus and a control.20250068-01 / 027644-8511

[0016] FIG. 26 illustrates an embodiment of the present method for analyzing a biological sample in a well.

[0017] The present teachings are best understood from the following detailed description when read with the accompanying drawing figures. The features are not necessarily drawn to scale. Wherever practical, like reference numerals refer to like features.DETAILED DESCRIPTION OF THE INVENTION

[0018] It is to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. The defined terms are in addition to the technical and scientific meanings of the defined terms as commonly understood and accepted in the technical field of the present teachings.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present teachings, some exemplary methods and materials are now described. All patents and publications referred to herein are expressly incorporated by reference.

[0020] As used herein, the terms “a,” “an,” and “the” include both singular and plural referents, unless the context clearly dictates otherwise. Thus, for example, “a well” includes one well and plural wells.

[0021] As used herein, and in addition to their ordinary meanings, the terms “substantial” or “substantially” mean to within acceptable limits or degree to one having ordinary skill in the art.20250068-01 / 027644-8511

[0022] The terms “approximately” and “about”, as used herein, mean to within an acceptable limit or amount to one having ordinary skill in the art. The term “about” generally refers to plus or minus 15% of the indicated number. For example, “about 10” may indicate a range of 8.5 to 11.5. For example, “approximately the same” means that one of ordinary skill in the art considers the items being compared to be the same. It should be understood that any of the values disclosed herein are also a disclosure of the approximate value (e.g., the disclosure of “0.10” shall also constitute a disclosure of “about 0.10”), and any disclosure of an approximate value is a disclosure of the value itself (e.g., the disclosure of “about 0.10” shall also constitute a disclosure of “0.10”), unless the context indicates otherwise. In the present disclosure, numeric ranges are inclusive of the numbers defining the range.

[0023] “Adjacent” as used herein means one element is next to, close to, or near another element. Adjacent can refer to elements that touch each other, are contiguous, or that share a structure or border, and / or to elements may or may not be in physical contact with each other.

[0024] An “analyte” as used herein means any substance undergoing analysis, such as cells, tissues, organelles, subcellular structures such as microtubules or cytoskeleton, molecules, or other components.

[0025] The term “liquid” as used herein means a material that generally conforms to the shape of its container while retaining a substantially constant volume independent of pressure. Liquid includes materials such as solutions, mixtures, dispersions, gels, suspensions and pastes.

[0026] The term “three-dimensional cell sample” as used herein means a sample that comprises cells at least in part in three dimensions (3D). A three-dimensional cell sample may include portions where the cells lie in two dimensions (e.g., a monolayers of cells), so long as another portion of the cell sample is in three dimensions. The term includes spheroids, islets,20250068-01 / 027644-8511 organoids, tissues, tissue slices, model organisms and cell aggregates. Cells analyzed with the present method and apparatus can be live cells, fixed cells, adherent cells, nonadherent cells, lysed cells, or mixtures thereof.

[0027] In some embodiments, the well surface or other element has a diameter or longest dimension across its area. More particularly, when the well surface or other element is circular, its longest dimension is its diameter; when the well surface is oval, its longest dimension is its major axis; when the well surface or other element is a square or rectangular, its longest dimension is its diagonal connecting opposite corners; for other shapes, its longest dimension can be determined in a manner consistent the foregoing examples.

[0028] Before the various embodiments are described, it is to be understood that the teachings of this disclosure are not limited to the particular embodiments described, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present teachings will be limited only by the appended claims.Apparatus For Metabolic Analysis Of Biological Samples

[0029] As one aspect of the present disclosure, an apparatus for metabolic analysis of a biological sample is provided. The present disclosure provides apparatus that solve one or more problems of existing analysis systems.

[0030] The present apparatus and methods improve sensitivity to allow for robust OCR and ECAR measurements in biological samples, including three-dimensional samples such as tissue slices and 3D models. In some embodiments, the present apparatus comprises an improved screen with larger openings that has been previously used, allowing for increased turnover of20250068-01 / 027644-8511 media and improved reagent diffusion to the sample. By way of example, a screen comprising a polyester mesh having a mesh opening of 285 pm is disclosed herein. The opening size and material of the screen can greatly reduce trapped bubbles, which often interfere with metabolic measurements, allowing for better well to well uniformity. It has also been found advantageous to decrease the ring height, creating a smaller microchamber which greatly increases the sensitivity. In some embodiments of the present methods, mixing parameters are improved to ensure sensor and microchamber recovery between measurements.

[0031] The present apparatus and methods solve one or more problems of prior approaches. For instance, prior approaches were unable to reproducibly measure mitochondrial respiration in tissue slices and other 3D models, or reliably measure ECAR with certain samples. Previous approaches also often captured bubbles, leading to unusable data. Tissue slices require proper medium turnover and perfusion of compounds. By changing the materials and dimensions of the mesh screen and decreasing the microchamber, one can now repeatably measure differences in basal OCR and ECAR in various tissue punch sizes. Additionally a more robust response is seen to reagents including FCCP (mitochondrial uncoupler), which were not able to be seen before. This will allow researchers to measure the parameters of mitochondrial respiration including basal respiration, ATP production, proton leak, spare capacity and non-mitochondrial oxygen consumption.

[0032] The present apparatus and methods are capable of measuring a distinct, significant difference in basal rates of tissue punches of various sizes, which could not be measured previously. One can accurately calculate spare respiratory capacity. Sample wells are not lost due to bubble interference. Sample is properly perfused, allowing for repeatable measurements. ECAR is no longer in the noise of the system, allowing for interrogation of glycolysis.20250068-01 / 027644-8511

[0033] In some embodiments, the present apparatus comprises a microplate having 24 wells with an inner-well geometry designed to contain cell samples comprising a three-dimensional matrix, for example cells embedded in a hydrogel. This microplate can be designed for compatibility with Seahorse XF analyzers or other devices for the measurement of cellular oxygen consumption and extracellular acidification.

[0034] The microplate comprises a frame having a frame surface and frame sides extending from the frame surface, and a plurality of wells. Each well has a top end, a bottom end opposite the top end, and one or more walls between the top end and the bottom end. Each well also has a well surface at the bottom end. The top end of each of the wells is surrounded by the frame and defines a top well opening. Each of the walls of the wells comprises a reservoir wall section adjacent to the bottom end, a tapered wall section adjacent to the reservoir wall section, and an upper wall section adjacent to the top end. In some embodiments, a second tapered wall section is between the (first) tapered wall section and the upper wall section. It is also contemplated that the walls can have additional wall sections, such as above or below the upper wall section and / or the tapered wall section(s).

[0035] FIG. 1 is an isometric view of an embodiment of a microplate 100 of the present apparatus having a frame 101 with frame sides 103 and a frame surface 105. The microplate 100 has twenty-four wells 102 having top ends with top well openings for receiving samples. The walls of each well can be circular, oval, square, rectangular, or another shape. In some embodiments, the walls have two, three, or more wall sections which differ from each other in their shapes or other properties. In some embodiments, the microplates 100 are configured for automated transport, incubation, and analysis in the imaging and analysis system. For example, the frame sides 103, frame surface 104, or other sections of the frame 101 can be configured to20250068-01 / 027644-8511 engage a robotic arm for automated transport or to fit within an incubator, plate reader, or storage environment.

[0036] FIG. 2 is a view of a bottom end of a well 102 which shows the wall sections of one of the well 102 more closely. The well 102 has a well surface 109, reservoir wall section 108, a shoulder section 110, a vertical wall section 111, and a tapered wall section 112. The well surface 109 is at the bottom end of the well. The reservoir wall section 108 can be perpendicular to the well surface 109 or can be at an angle (as illustrated in FIG. 2). In some embodiments, the well surface can have a structure or composition that enhances cell attachment, supports cell proliferation, promotes formation of a three dimensional cell sample, or otherwise facilitates cell culture. In some embodiments, the well surface has a coating disposed thereon.

[0037] Al is an angle of a reservoir wall section 108 relative to well surface 109 or to an axis parallel to the well surface. In some embodiments, angle Al is about 90 degrees, or at least 90 degrees, or at least 95 degrees, or at least 100 degrees, or at least 105 degrees, or at least 110 degrees, or at least 115 degrees, or at least 120 degrees, or at least 135 degrees; alternatively or additionally, angle Al is at most 160 degrees, or at most 150 degrees, or at most 140 degrees, or at most 135 degrees, or at most 130 degrees, or at most 125 degrees, or at most 120 degrees, or at most 115 degrees, or at most 110 degrees, or at most 105 degrees, or at most 100 degrees. It is contemplated that any of the foregoing minimums and maximums can be combined to form a range for angle Al, so long as the minimum is smaller than the maximum (e.g., from 90 to 120 degrees).

[0038] Hl is a height of a reservoir wall section 108, and W1 is a width or longest dimension of the well surface 109 at the bottom end 106 of the well 102. In some embodiments, the height (Hl) of a reservoir wall section 108 is at least 0.1 mm, or at least 0.25 mm, or at least 0.5 mm, or20250068-01 / 027644-8511 at least 0.6 mm, or at least 0.7 mm, or at least 0.75 mm, or at least 0.8 mm, or at least 0.9 mm, or at least 0.95 mm, or at least 1 mm; alternatively or additionally, the height (Hl) of a reservoir wall section 108 is at most 4 mm, or at most 3 mm, or at most 2.5 mm, or at most 2 mm, or at most 1.75 mm, or at most 1.5 mm, or at most 1.4 mm, or at most 1.3 mm, or at most 1.25 mm, or at most 1.2 mm, or at most 1.1 mm, or at most 1.05 mm, or at most 1 mm, or at most 0.95 mm, or at most 0.9 mm, or at most 0.8 mm, or at most 0.75 mm, or at most 0.7 mm, or at most 0.5 mm. It is contemplated that any of the foregoing minimums and maximums can be combined to form a range for height (Hl), so long as the minimum is smaller than the maximum (e.g., from 0.5 mm to 2 mm).

[0039] In some embodiments, the W1 is a width (or longest dimension) of the well surface 109 is at least 0.5 mm, or at least 1 mm, or at least 1.5 mm, or at least 2 mm, or at least 2.5 mm, or at least 3 mm, or at least 3.25 mm, or at least 3.5 mm, or at least 3.75 mm, or at least 4 mm, or at least 4.5 mm, or at least 5 mm, or at least 6 mm; alternatively or additionally, the width or longest dimension (Wl) of a well surface 109 is at most 16 mm, or at most 12 mm, or at most 10 mm, or at most 9 mm, or at most 8 mm, or at most 7 mm, or at most 6.5 mm, or at most 6 mm, or at most 5.5 mm, or at most 5 mm, or at most 4.5 mm, or at most 4.25 mm, or at most 4 mm, or at most 3.75, or at most 3.5 mm, or at most 2 mm. It is contemplated that any of the foregoing minimums and maximums can be combined to form a range for width or longest dimension (Wl), so long as the minimum is smaller than the maximum (e.g., from 1.5 mm to 6 mm).

[0040] W2 is a width of the shoulder section 110, that is, the width of the wall section having a doughnut shape with an opening to the reservoir. In some embodiments, the width (W2) of the well opening is at least 0.1 mm, or at least 0.25 mm; or at least 0.5 mm; or at least 0.7 mm, or at least 0.8 mm, or at least 0.9 mm, or at least 0.95 mm, or at least 1 mm; or at least 1.1 mm, or at20250068-01 / 027644-8511 least 1.5 mm, or at least 2 mm, or at least 3 mm; alternatively or additionally, the width (W2) of the well opening is at most 6 mm, or at most 4 mm, or at most 2 mm, or at most 1.5 mm, or at most 1.3 mm, or at most 1.2 mm, or at most 1.1 mm, or at most 1.05 mm, or at most 1 mm, or at most 0.9 mm, or at most 0.75 mm. It is contemplated that any of the foregoing minimums and maximums can be combined to form a range for width or longest dimension (W2), so long as the minimum is smaller than the maximum (e.g., from 0.5 mm to 2 mm).

[0041] In some embodiments, the shoulder section 110 and / or the vertical wall section 111 may comprise one or more centering features configured to center a probe (e.g. a plunger) to be substantially concentric with the reservoir wall section. For example, the centering feature can comprise a ramped rib (not shown), or series of ramped ribs that extend that extend radially outward from a substantially central location in the well, with a gradual slope upward toward shoulder wall section 110.

[0042] In some embodiments, the wall(s) of the wells (or one or more wall section(s)) are completely or partially opaque, in that they block transmission of light. More particularly, the upper wall section, and / or the tapered wall section, and / or the shoulder section, and / or the reservoir wall section can be opaque. The microplate can be formed from an opaque material, or the wall(s) of the wells can be painted or coated to render them opaque.

[0043] The microplate can be formed from any suitable material which does not affect measurement of a sample and which has appropriate properties for manufacturing and use.Examples of materials include polyolefin-based or cyclic polyolefin-based resins such as norbomene polymers and copolymers, tetracyclodecene polymers and copolymers, and other polyolefin copolymers; polyethylene, polypropylene, ethyl ene-propylene copolymer; polystyrene-based resins such as polystyrene and acrylonitrile-butadiene-styrene-based resins;20250068-01 / 027644-8511 (meth)acrylic resins such as acrylic polymethacrylates, polymethylmethacrylates, and polyacrylonitriles, polycarbonate resins; polyethylene terephthalate resins; vinyl chloride resins; polybutylene terephthalate resins; polyacrylate resins; polysulfide resins; polyethersulfone resins; polyetheretherketone resins; polyetherimide resins; fluorine-based resins such as polytetrafluoroethylenes; polymethylpentene resin; and cellulosic resins.

[0044] In some embodiments, the reservoir wall section 108 has a height which is from about O.Olx to about 2x of the diameter or longest dimension of the well surface. For example, for a well having a circular well surface of 4 mm and a reservoir wall section of 1 mm, the well has a reservoir wall section height that is 0.25 of the well surface diameter. In some embodiments, the ratio of reservoir wall section height to well surface longest dimension can be a minimum of 0.01, 0.02, 0.05, 0.1, 0.15, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40 or 0.5. In some embodiments, the ratio of reservoir wall section height to well surface longest dimension can be a maximum of 2, 1.5, 1, 0.75, 0.5, 0.33, 0.3, 0.29, 0.28, 0.27, 0.20, 0.25, 0.24, 0.23, 0.22, 0.21, 0.2, 0.15, or 0.10. It is contemplated that any of the foregoing minimums and maximums can be combined to form a range for the ratio, so long as the minimum is smaller than the maximum (e.g., from 0.1 to 0.5).

[0045] The microplates of the present technology can be configured in any manner or orientation including having dimensions that are consistent with the well number and spacing of standard microplates. For example, in some embodiments, a microplate of the present technology comprises at least 6 or at least 8 wells. In other embodiments, a microplate of the present technology comprises at least 24 wells. In some embodiments, a microplate of the present technology comprises at least 48 wells, or at least 96 wells, or at least 384 wells, or at least 1536 wells.20250068-01 / 027644-8511

[0046] FIG. 3 shows an embodiment of a ring comprising a screen for the present apparatus. Ring 314 comprises a screen 316 attached thereto. The ring 314 comprises a screen 316 configured to cover the reservoir. In some embodiments, the screen has openings larger than 170 pm, alternatively 180 pm or larger, alternatively 200 pm or larger, alternatively 220 pm or larger, alternatively 250 pm or larger, alternatively 285 pm; alternatively or additionally, the screen has openings smaller than 1 mm, or at most 750 pm, or at most 500 pm, or at most 350 pm. It is contemplated that any of the foregoing minimums and maximums can be combined to form a range for the screen openings, so long as the minimum is smaller than the maximum (e.g., from 250 pm to 350 pm). In some embodiments, the openings are substantially square, and the size of the opening refers to the size of the side of the square. When the openings in the screen are circular, the size refers to its diameter. When the openings are oval or rectangular, the size generally refers to their longest dimensions. The size of openings of a screen can be identified in a manner consistent with the examples herein or in accordance with common usage in the art.

[0047] In some embodiments, the screen has an open area less than 55%, alternatively 54% or less, alternatively 53% or less, alternatively 52% or less, alternatively 51% or less, alternatively 50% or less, alternatively 49% or less, alternatively 48% or less, alternatively 47% or less, alternatively 47% or less, alternatively 45% or less, alternatively 44% or less; alternatively or additionally, the screen has an open area more than 30%, alternatively more than 35%, more than 40%. It is contemplated that any of the foregoing minimums and maximums can be combined to form a range for the screen open area, so long as the minimum is smaller than the maximum (e.g., from 30% to 50%). Open area of a screen generally refers to area that is not occupied by physical structures (e.g., fibers or membrane) of a screen. The percentage of open20250068-01 / 027644-8511 area of a screen can be identified in a manner consistent the examples herein or in accordance with common usage in the art.

[0048] In some embodiments, the screen is a mesh (such as interwoven threads, fibers or microfibers), a porous frit (such as a glass or ceramic frit), a porous membrane (such as a polymer membrane). In some embodiments, the screen comprises monofilament woven fibers. In some embodiments, the screen comprises polyester fibers.

[0049] In some embodiments, the fibers have a hydrophilic coating. In some embodiments, the screen is hydrophilic, such as by coating or by being formed from a hydrophilic material.

[0050] T1 is the thickness of a screen 316. In some embodiments, the thickness (Tl) of the screen 316 is at least 0.02 mm, or at least 0.04 mm, or at least 0.06 mm, or at least 0.08 mm, or at least 0.1 mm, or at least 0.125 mm, or at least 0.15 mm, or at least 0.16 mm, or at least 0.17 mm, or at least 0.18 mm, or at least 0.19 mm, or at least 0.2 mm, or at least 0.21 mm, or at least 0.22 mm, or at least 0.23 mm, or at least 0.24 mm, or at least 0.25 mm, or at least 0.275 mm, or at least 0.3 mm, or at least 0.35 mm, or at least 0.4 mm, or at least 0.5 mm, or at least 0.6 mm; alternatively or additionally, the thickness (Tl) of the screen 316 is at most 1 mm, or at most 0.9 mm, or at most 0.8 mm, or at most 0.7 mm, or at most 0.6 mm, or at most 0.5 mm, or at most 0.4 mm, or at most 0.3 mm, or at most 0.29 mm, or at most 0.28 mm, or at most 0.27 mm, or at most 0.26 mm, or at most 0.25 mm, or at most 0.24 mm, or at most 0.23 mm, or at most 0.22 mm, or at most 0.21 mm, at most 0.2 mm, or at most 0.19 mm, or at most 0.18 mm, or at most 0.17 mm, or at most 0.16 mm, or at most 0.15 mm, or at most 0.125 mm, or at most 0.1 mm. It is contemplated that any of the foregoing minimums and maximums can be combined to form a range for thickness (Tl), so long as the minimum is smaller than the maximum (e.g., from 0.1 mm to 0.3 mm).20250068-01 / 027644-8511

[0051] In some embodiments, the ring has a ring width (W3) equal to or less than a width of the shoulder section, such that when the ring is placed on the shoulder section, the screen covers the reservoir. W3 is a width of ring 314, that is, the dimension of the wall of ring 314 in a horizontal direction. W3 can be equivalent to W2, or larger or smaller. In some embodiments, the width (W3) of the ring 314 is at least 0.1 mm, or at least 0.25 mm; or at least 0.5 mm; or at least 0.7 mm, or at least 0.8 mm, or at least 0.9 mm, or at least 0.95 mm, or at least 1 mm; or at least 1.1 mm, or at least 1.5 mm, or at least 2 mm, or at least 3 mm; alternatively or additionally, the width (W3) of the ring 314 is at most 6 mm, or at most 4 mm, or at most 2 mm, or at most 1.5 mm, or at most 1.3 mm, or at most 1.2 mm, or at most 1.1 mm, or at most 1.05 mm, or at most 1 mm, or at most 0.9 mm, or at most 0.75 mm. It is contemplated that any of the foregoing minimums and maximums can be combined to form a range for width or longest dimension (W3), so long as the minimum is smaller than the maximum (e.g., from 0.5 mm to 2 mm).

[0052] In some embodiments, the ring has a ring height (H2), that is, a dimension of the wall of the ring in a vertical direction. H2 (shown in FIG. 5) is a height of ring, In some embodiments, ring height (H2) is less than 1.17 mm, alternatively 1 mm or less, alternatively 0.75 or less, alternatively 0.65 or less, alternatively 0.58 mm or less; alternatively or additionally, ring height (H2) can be 0.1 mm or more, alternatively 0.25 mm or more, alternatively 0.5 mm or more. It is contemplated that any of the foregoing minimums and maximums can be combined to form a range for ring height (H2), so long as the minimum is smaller than the maximum (e.g., from 0.25 mm to 1 mm).

[0053] In some embodiments, a screen material is thermally bonded to ring and cut with a dye cut. In some embodiments, screen 316 is attached to one side of ring 314. In some embodiments, screen 316 is sandwiched between separate sections of ring 314. In some20250068-01 / 027644-8511 embodiments, the microplate is formed by introducing a moldable material into a molding device. The moldable material can be a thermoplastic that solidifies by cooling, or a thermosetting material that solidifies by curing. Various polymers can be used as the moldable material for the manufacture of the microplate, such as cyclic olefin polymer or copolymer, polystyrene (PS), polyethylene (PE) or polypropylene (PP). The moldable material after solidification should be generally inert to samples or reagents typically encountered in cell culture or analysis, resistant to the absorption of biomolecules, and able withstand expected temperatures. The microplates can then be tissue culture treated and gamma-irradiated prior to providing them to an end user.

[0054] FIG. 4 illustrates an embodiment of the present apparatus where a ring 414 comprising a screen 416 is positioned in a well 402. The ring 414 is positioned at a bottom end of the well 402, generally within the vertical wall sections (not shown) beneath tapered wall section 412.Analysis Systems Comprising The Present Apparatus

[0055] As another aspect of the present disclosure, analysis systems comprising the present apparatus are provided. The analysis systems comprise a microplate and a ring as described herein. The systems also comprise a device for obtaining metabolic measurements. Exemplary devices for use with the present apparatus in an analysis system include Seahorse XF instruments. Additional details about devices for obtaining metabolic measurements can be found in Teich et al. US Patent 9,170,253; McGarr et al. US Patent 9,494,577; and Taber US Pat. App. Pub. 20240159672A1.20250068-01 / 027644-8511

[0056] In some embodiments, the device for obtaining metabolic measurements comprises one or more plungers adapted for movement within the wells. The plunger and well, especially the vertical wall section of the well, are configured and dimensioned to fit together preferably so that insertion and reciprocation of the plunger into the well permits agitating and mixing of the liquid medium. The plunger and the well are configured to induce perfusion as the plunger moves downwardly or upwardly and liquid medium contacts the biological sample. The flow of liquid medium induced by movement of the plunger permits exposure of the biological sample to fresh liquid medium. In some embodiments, the device defines a 0.2 inches plunger stroke and / or defines a stroke speed of about 0.16 inch / second.

[0057] In a preferred embodiment, sensors may be disposed on the plungers for detecting the concentration of analytes in a liquid medium in contact with the biological sample. The sensors may measure the concentration of oxygen, carbon dioxide, and / or hydrogen ions dissolved in media about the sample.

[0058] FIG. 5 is a cross-sectional view of one embodiment of the present apparatus having a biological sample contained in a well 502 of the present well plate in association with a device for obtaining metabolic measurements. The figures are for illustration and their features are not drawn to scale. FIG. 5 shows a well 502 of a microplate 500 having a biological sample 520 in a liquid medium. The sample 520 is in a reservoir 507 of the well 502, and a screen 516 attached to a ring 514 is above the sample 520. A plunger 522 is lowered into well 502, through tapered wall section 512 and within vertical wall section 511. A reservoir 507 is formed by the reservoir wall section 508 and the well surface 509. The reservoir 507 contains a biological sample 520, such as a three-dimensional cell sample, contained in a liquid medium that serves to hydrate the gel and submerge the distal end of the probe 522. In some embodiments, the biological sample20250068-01 / 027644-8511 comprises cells and a matrix medium such as a hydrogel. The sample 520 contains one or more analytes or metabolic parameters to be tested. In FIG. 5, the plunger 522 is shown in association with the reservoir such that the plunger 522 is in contact with or close proximity to the ring 514.

[0059] Plunger 522 has a sensor 524 for measuring one or more metabolic parameters. In some embodiments, sensor 524 is a solid state sensor or other material deposited onto the plunger 522 that senses luminescence. For example, sensor 524 can comprise a fluorophore in a solid material. The plunger can be configured as an optical wave guide where a signal source (typically and LED or laser) can be used to excite the fluorophore sensor through the pillar (wave guide). The plunger 522 can be configured to contain or receive fiberoptics, glass, open air, etc., to facilitate optical transmission to and from the fluorophore. Parameters from the fluorophore such as intensity, phase, time decay, or combinations of the foregoing or other parameters can be used to measure an analyte or an analyte’s concentration. The emission signal from the fluorophore signal can be measured or detected by an electro-optical or optical sensor such as cameras, photodiodes, multi-pixel photon counters (MPPC), photomultiplier tubes (PMT), time-of-flight (TOF) sensors, combination of the foregoing and combinations with other sensors). Alternatively, the present methods and apparatus can comprise contacting one or more fluorescent emission or phosphorescent emission agents, such as fluorophores, with the sample 520 or the liquid medium that are influenced by the presence of a biological analyte when undergoing a fluorescent emission or phosphorescent emission.

[0060] The cross-sectional view of the device shows a plunger 524 of the device 530 lowered into the well 502, and it is contemplated that one or more metabolic measurements can be obtained while plunger 524 is in that position. It is also contemplated that the plunger 524 can move downward or upward before, during or after metabolic measurements are obtained. In20250068-01 / 027644-8511 some embodiments, metabolic measurements are obtained in a method that comprises a plunger stroke of from 0.05 inch to 0.4 inch, alternatively about 0.2 inch, and / or defines a stroke speed of from 0.04 inch / second to 0.32 inch / second, alternatively about 0.16 inch / second.

[0061] The plunger 522 is adapted to reciprocate between a lowered testing position, as shown in FIG. 5, and a raised waiting position where the plunger is withdrawn from the area of the vertical wall Section 511. Lowering of the plunger 522 can be guided by the vertical wall section 511 or by other centering features such as ribs where the wall section is wider than the plunger 522. As illustrated in FIG. 5, the plunger 522 is adapted for lowering into the well 502 so as to form a measurement volume defined by the plunger 502, the reservoir wall section 502, the well surface 509, and the interior of the ring 514.

[0062] In some embodiments, the device 530 comprises a cartridge that also includes one or more ports adapted to deliver fluids such as liquids and gases into the well 502. For example, in one embodiment, the ports can be in communication with an external gas supply and an internal air control. The external gas supply and the internal air control can control gases fed to or removed from the head space above the liquid medium.

[0063] In addition to serving to reduce the volume for measurement, the plunger 502 can also serve as a probe. Plunger 522 can include fiber optics that deliver excitation light to the biological sample 520 and transmit fluorescent emissions or phosphorescent emissions to an electrooptic sensor or other photodetection sensor.

[0064] In some embodiments, the analysis system further comprises a heater. In some embodiments, the heater contacts the walls of the wells, and the exteriors of the walls of the well are configured for engaging the heater.20250068-01 / 027644-8511

[0065] In some embodiments, the analysis system further comprises one or more controllers in signal communication with (by wired or wireless means) the microplate 500 or the light the device 530 for obtaining metabolic measurements, or other components that hold or support them (such as a plate holder, a robotic arm, a gantry, or other actuator). The one or more controllers comprise, or are configured to receive and implement, one or more software programs for determining positions of the wells relative to the light source 210, the microscopic imager 220, and / or the device 230, and for moving one or more components to facilitate the imaging and / or analysis of the biological samples within the wells. The controller 240 may be, for example, a desktop computer, laptop computer, portable computer, tablet computer, handheld computer, mobile computing device, personal digital assistant (PDA), smartphone, etc., and may include one or more types of hardware (e.g., electronic-based processors, memories, user input peripherals, user output peripherals, etc.), firmware, and / or software. The imaging system may also include other electronics (not shown) configured for controlling movement or operation of the various components.Methods of Analyzing Three Dimensional Biological Samples

[0066] As another aspect of the present disclosure, methods are provided for analyzing biological samples. The methods can comprise providing a biological sample into one or more of the wells of an embodiment of the present microplates. The biological sample can be placed in the reservoir of the well or placed onto a face of the screen. A metabolic property or parameter of the three-dimensional cell sample is measured, and / or an image is obtained of the biological sample.

[0067] The present apparatus can be used to obtain measurements of biological samples, such as three dimensional cell samples or portions thereof such as cells, tissues, organelles,20250068-01 / 027644-8511 subcellular structures such as microtubules or cytoskeleton, molecules, or other components. In some embodiments, the methods comprise obtaining metabolic measurements of live cells, fixed cells, adherent cells, nonadherent cells, lysed cells, or mixtures thereof. In some embodiments, the methods comprise obtaining metabolic measurements of live tissue, tissue slices, tissue punches, spheroids, islets, organoids, tissues, tissue slices, model organisms, cell aggregates, or mixtures thereof . In some embodiments, the biological sample is a tissue obtained from a mammalian subject, such as a human, non-human primate, rodent, canine, or other. In some embodiments, the biological sample is a tissue slice or tissue punch from an organ or tumor, such as heart tissue, brain tissue, liver tissue. The microplates can also be used for live cell imaging, fixation techniques, immunostaining, and standard fluorescence detection assays of biological samples.

[0068] In some embodiments, the present methods comprise adding a biological sample to the wells of the microplate. In some embodiments, the methods comprise adding a live tissue obtained from a subject to the wells. In some embodiments, the methods comprise obtaining a sample of a live tissue from a subject, such as by dissecting or performing a biopsy. In some embodiments, the methods comprise preparing a tissue slice or a tissue punch from the sample of live tissue, adding the tissue slice or tissue punch to a reservoir of the well, and placing a ring comprising a screen in the well such that the screen covers the reservoir. In some embodiments, the biological sample is a tissue punch having a diameter from 1 mm to 3 mm, alternatively from 1.5 mm to 2.5 mm, alternatively about 2 mm.

[0069] In some embodiments, the biological sample comprises one or more cells. A cell composition comprising the cells (such as cells in a matrix medium such as a hydrogel) can be added by pipetting, injecting, or otherwise depositing into the well. In some embodiments, the20250068-01 / 027644-8511 composition has a volume of 300 pL or less, alternatively of 250 pL or less, alternatively of 200 pL or less, alternatively of 150 pL or less, alternatively of 100 pL or less, alternatively 50 pL or less, alternatively of 40 pL or less, alternatively of 30 pL or less, alternatively of 20 pL or less, alternatively of 10 pL or less.

[0070] Suitable hydrogels for culturing three-dimensional cell samples are permeable to dissolved oxygen, nutrients and metabolites of cells. Hydrogel can be an extracellular matrix based on the basement membrane of animal cells and can comprise laminin, collagen, fibronectin, proteoglycans, and / or polymers such as polyethylene glycol (PEG). Commercially available hydrogels include Coming™ Matrigel™ Matrix.

[0071] In some embodiments, the cells added to the wells are incubated for an incubation period before an image is obtained to form three-dimensional cell samples, such as spheroids, islets, or organoids. In some embodiments, the microplates seeded with cells can be moved to an incubator to incubate the cells. For example, a microplate seeded with cells can be incubated for cell growth or expansion.

[0072] In some embodiments, a user suspends a cell culture in a hydrogel and pipettes up to a specified volume within the reservoir section (e.g. up to 12 pL for a 1mm x 4mm reservoir section) of the mixture into the inner column of each well such that the upper level of the hydrogel leaves some space (e.g. 5% of the height) between the hydrogel and the sensors 335 of the probe 331. As the cell culture proliferates and (where appropriate) differentiates, organoids are formed within or on the gel. After a desired culturing period, the plates can be measured using a device for obtaining metabolic measurements, such as an XF instrument, to generate oxygen consumption and extracellular acidification results for the user.20250068-01 / 027644-8511

[0073] In some embodiments the present methods also comprise obtaining one or more images of the biological samples using an imaging microscope. In some embodiments, the present methods comprise contacting the biological sample or an analyte in the liquid medium with a label before obtaining the image. Examples of labels include chromophores, fluorophores, phosphors, quantum dots, luminescent or chemiluminescent molecules, radioisotopes, magnetic nanoparticles, and mass tags.

[0074] The present methods can comprise obtaining one or more images of the three-dimensional cell samples in the wells. The images can be viewed while being obtained and / or recorded for later viewing. In some embodiments, the methods and apparatus obtain brightfield images, color brightfield images, phase contrast images, fluorescent images, phosphorescent images, and / or images from other modalities.

[0075] The present methods can further comprise analysis of the biological sample or the obtained images of the biological samples, such as by analysis of cellular growth rate, cell morphology, nuclear morphology, cellular responses (such as to drugs or other stimuli), protein expression (such as receptor expression or cytokine expression), mRNA expression, metabolic properties, pH levels or changes, cellular waste products (such as carbon dioxide, water, lactic acid, urea, or ammonia), or a combination thereof. In some embodiments, cells from the microplate have their nucleic acids (DNA and / or RNA) sequenced and analyzed.

[0076] In some embodiments, the present methods and apparatus are used for metabolic measurements. For example, they can be used for metabolic measurements of cells in a culture, such as overall cellular metabolic and respirative rates, mitochondrial respiration, measurements of glycolysis, adenosine triphosphate (ATP) production, relative proportion of aerobic to anaerobic respiration, relative rates of consumption of various metabolic substrates, effect of20250068-01 / 027644-8511 stimulation of certain transmembrane and other cellular receptors, rates of production of various secreted factors, and cell viability. In some embodiments, the metabolic measurements are OCR and / or ECAR. Further details about measuring extracellular acidification and oxygen consumption rates can be found in Neilson et al. US Pat. App. Publication No. 20070087401A1, Neilson et al. US Patent No. 8,202,702, Ferrick et al. US Pat. App. Publication No.20160281049A1, each of which is incorporated herein by reference.

[0077] In some embodiments of the present methods, a metabolic potential of the biological sample may be determined by (i) providing the initial oxygen consumption rate, the initial extracellular acidification rate, the subsequent oxygen consumption rate, and the subsequent extracellular acidification rate to a software program, and (ii) using the software program to calculate the metabolic potential. Data analysis begins by determining the baseline rate of oxygen consumption and extracellular acidification as defined by the last measurement prior to injection of stressors. The stressed data point is determined using the data point which is stimulated maximally following co-injection of oligomycin and FCCP. These data are plotted graphically where the Y-axis represents OCR and the X axis represents ECAR. Metabolic potential is determined for each element separately by determining the percent stimulation of each pathway (OCR and ECAR) by the stressor above baseline.

[0078] In some embodiments, the present methods also include forming a measurement volume by lowering a plunger to contact the shoulder section, so that the volume is reduced. The reduced volume can be from about 0.5% to about 50% of the original volume of the well, alternatively from about 5% to about 20% of the original volume, alternatively less than about 5% of the original volume.20250068-01 / 027644-8511

[0079] In some embodiments, one or more sensors can be used to measure metabolic parameters of the biological sample in the wells of the imaging wall plate. In some embodiments, the sensor(s) are adapted for measuring one or more analytes in a liquid medium in contact with the biological sample. The sensors can be a fluorescent sensor, a luminescent sensor, an ISFET sensor, a surface plasmon resonance sensor, a sensor based on an optical diffraction principle, a sensor based on a principle of Wood’s anomaly, an acoustic sensor, or a microwave sensor. The present technology is not limited to any particular cellular assays, measurements or sensors. In some embodiments, the microplates are used in conjunction with an optical fiber probe, such as probes used in the Agilent XF instruments.

[0080] The present methods and apparatus can be used in a variety of fields related to cell culture and analysis. Such fields include, but are not limited to, biological research, drug discovery, and clinical diagnostics. For example, as a drug discovery tool, the microplates or imaging systems can be used to screen various molecules for an effect on cellular metabolism in co-culture, protein secretion, or intra / extra cellular ion exchange. The present methods and apparatus can also be used to determine the health of a biological sample in culture, including co-culture, both before and after a conventional assay is performed, thereby improving the performance of such an assay.Exemplary Embodiments

[0081] Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, those set forth in the claims, description, and examples. Examples

[0082] Several experiments were performed using the present apparatus in comparison to a control which used a currently available insert comprising a ring and screen with a microplate.20250068-01 / 027644-8511 The experiments used metabolic measurements of biological samples in the apparatus and control to assess their capabilities to obtain such measurements from three-dimensional biological samples. The metabolic measurements were obtained using a Seahorse Bioscience XF24 Extracellular Flux Analyzer.

[0083] For the experimental tests, a black ring having a height of 0.58 mm and a screen having a mesh opening of 285 pm and open area of 44% was used. For the controls, the white ring had a height of 1.17 mm and the screen had a mesh opening of 170 pm and an open area of 55%.

[0084] Various types of biological samples were placed into reservoirs of wells of microplates. The ring and screen were positioned into the wells so as to contact a shoulder section. A liquid medium was provided to the wells. Baseline measurements of cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were performed.

[0085] Oligomycin was added to each well, followed by measurement of OCR and ECAR. Oligomycin blocks the ATPase of complex V, thus preventing the hydrolysis of adenosine triphosphate (“ATP”), that would supply electrons through this complex in the reverse direction which would counteract the effects of FCCP. Oligomycin also “stresses” the glycolytic system to produce more ATP to counteract the loss of ATP caused by its inhibition of the mitochondrial ATPase. OCR was then plotted as in the example figure to determine the lowest concentration which achieves maximal stimulation of OCR.

[0086] A prototypic uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (“FCCP”) was added to each well, followed by measurement of OCR and ECAR. FCCP stresses the aerobic system by dissipating the proton gradient across the inner mitochondrial membrane. This induces mitochondrial uncoupling and causes cells to increase their metabolic rate and20250068-01 / 027644-8511 therefore OCR and ECAR, and forces the biological sample to increase mitochondrial activity up to its maximum.

[0087] A Complex I inhibitor, rotenone, is added to the samples. This will shut down mitochondrial respiration and enable both the mitochondrial and non-mitochondrial fractions contributing to respiration to be calculated. One will observe a decrease in OCR due to impaired mitochondrial function, with a concomitant increase in ECAR as the cell shifts to a more glycolytic state in order to maintain its energy balance.Example 1

[0088] Metabolic measurements were obtained for brain tissue samples. For the control, bubbles were present under the insert, a delay in ECAR level data was observed (as shown in FIG. 6A), and OCR rates were lower (as shown in FIG. 6B). For the present apparatus, no bubbles were under the screen, no delay in ECAR level data was observed (as shown in FIG. 7A), and OCR rates were higher (as shown in FIG. 7B). Basal OCR CV% and outliers were both lower using the present apparatus than with the control.

[0089] Data obtained from the experiment were recalculated and the results are shown in FIGS. 8A to 9B. FIGS. 8A and 8B show data for the control, and FIGS. 9A and 9B show data using the present apparatus.

[0090] FIGS. 10A and 10B show OCR and ECR measurements from the experiment using the present apparatus. FIGS. 11 A and 1 IB show OCR and ECR measurements from the experiment using the control.

[0091] Data obtained from the experiment were recalculated and the results are shown in FIGS. 12Ato 13B. FIGS. 12A and 12B show OCR and ECR measurements from the experiment20250068-01 / 027644-8511 using the present apparatus. FIGS. 13 A and 13B: control show OCR and ECR measurements from the experiment using the control.Example 2

[0092] Metabolic measurements were obtained for liver tissue samples. FIGS. 14, 16, and 18 show liver OCR data obtained from three microplates (plates 2, 3, 4) using the present apparatus. FIGS. 15, 17, and 19 show liver OCR data obtained from three microplates (plates 2, 3, 4) using the control. FIGS. 20, 22 and 24 show liver ECAR data obtained from three microplates (plates 2, 3, 4) using the present apparatus. FIGS. 21, 23 and 25 show liver ECAR data obtained from three microplates (plates 2, 3, 4) using the control.Example 3

[0093] In this example, an embodiment of the present method for analyzing a biological sample in a well is provided, where the biological sample is a tissue obtained from a mammal (such as heart tissue, brain tissue, or liver tissue). FIG. 26 illustrates the method. Tissue is freshly dissected and transferred to appropriate chilled buffer solution. Tissue is sliced, on ice, to desired thickness and transferred to a punch pad using a brush or forceps. Biopsy punches are used to create a uniform sample between 1mm and 3mm. Punches are then placed in the reservoir of a microplate like that shown in FIG. 2. A ring like that of FIG. 3 is then installed into each well using either forceps or the insert tool. Assay media is slowly added to each well, taking care to not introduce bubbles. The microplate is placed in an incubator without CO2 at a temperature of 37°C for Ihr prior loading on a XF Flex instrument for metabolic measurement. During this incubation period, desired compounds are prepared and loaded into the XF cartridge ports. The20250068-01 / 027644-8511 cartridge is loaded into the XF Flex instrument for calibration. When calibration is completed, the microplate containing the tissue samples is loaded for analysis.Example 4

[0094] In this example, an embodiment of the present apparatus and method for analyzing a biological sample in a well is provided. The example is set forth in Appendix A, which is incorporated herein by reference.Example 5

[0095] In this example, an embodiment of the present apparatus and method for analyzing a biological sample in a well is provided. The example is set forth in Appendix B, which is incorporated herein by reference.

Claims

20250068-01 / 027644-8511CLAIMSWe claim:

1. An apparatus for metabolic analysis of biological samples comprising:(a) a microplate comprising a plurality of wells, wherein each of the wells has a top end, a bottom end opposite the top end, a well surface at the bottom end, and one or more walls between the top end and the bottom end, wherein each of said one or more walls comprises:(i) a reservoir wall section adjacent to the bottom end, wherein the reservoir wall section and the well surface form a reservoir;(ii) a shoulder section adjacent to the reservoir wall section; and (iii) a vertical wall section adjacent to the shoulder section;(b) a ring comprising a screen configured to cover the reservoir, wherein the screen has openings larger than 170 pm, alternatively larger than 200 pm, alternatively larger than 250 pm, alternatively 285 pm.

2. The apparatus of claim 1, wherein the screen has open area less than 55%, alternatively 50% or less, alternatively 44% or less.

3. The apparatus of claim 1 or 2, wherein the screen is a mesh, a porous frit, or a porous membrane.

4. The apparatus of any of claims 1 to 3, wherein the screen comprises monofilament woven fibers.20250068-01 / 027644-8511 5. The apparatus of any of claims 1 to 4, wherein the screen comprises polyester fibers.

6. The apparatus of claim 5, wherein the polyester fibers form a mesh.

7. The apparatus of claim 5, wherein the fibers have a hydrophilic coating.

8. The apparatus of any of claims 1 to 7, wherein the screen is hydrophilic.

9. The apparatus of any of claims 1 to 8, the ring has a ring thickness equal to or less than a width of the shoulder section, such that when the ring is placed on the shoulder section, the screen covers the reservoir.

10. The apparatus of any of claims 1 to 9, the ring has a ring height less than 1.17 mm, alternatively 1 mm or less, alternative 0.75 or less, alternatively 0.58 mm or less.

11. The apparatus of any of claims 1 to 10, the ring is opaque.

12. The apparatus of any of claims 1 to 11, the ring is polyethylene terephthalate (PET).20250068-01 / 027644-8511 13. The apparatus of any of claims 1 to 12, wherein the well surface has a diameter or a longest dimension of from about 1 mm to about 8 mm, alternatively from about 2 mm to about 4 mm, alternatively about 3.15 mm.

14. The apparatus of any of claims 1 to 13, wherein the reservoir wall section has a height from about 50 pm to about 1000 pm, alternatively from about 100 pm to about 400 pm, alternatively from about 250 pm.

15. The apparatus of any of claims 1 to 14, wherein some or all of the one or more walls of the microplate is opaque.

16. The apparatus of any of claims 1 to 15, wherein the microplate further comprises a tapered wall section adjacent to the vertical wall section.

17. The apparatus of any of claims 1 to 16, wherein the microplate is formed from a thermoplastic material.

18. The apparatus of claim 17, wherein the thermoplastic material comprises polystyrene, polyethylene terephthalate (PET), polypropylene, polycarbonate, a cyclic olein polymer and / or a cyclic olefin copolymer.

19. The apparatus of any of claims 1 to 18, wherein the well surface is covered by a coating.20250068-01 / 027644-851120. A method of analyzing a biological sample in a well, the method comprising: providing a biological sample into one or more of the wells of the apparatus of any one of claims 1 to 19; andobtaining a metabolic measurement of the biological sample.

21. The method of claim 20, wherein the biological sample is a three-dimensional cell sample.

22. The method of claim 20, wherein the biological sample is a live tissue.

23. The method of claim 22, wherein biological sample is a tissue slice or tissue punch.

24. The method of any of claims 20 to 23, wherein the biological sample is a heart tissue.

25. The method of any of claims 20 to 23, wherein the biological sample is a brain tissue.

26. The method of any of claims 20 to 23, wherein the biological sample is a liver tissue.20250068-01 / 027644-8511 27. The method of claim 20, wherein the biological sample is provided by adding one or more cells to the one or more wells, and expanding the one or more cells.

28. The method of any of claims 20 to 27, wherein the biological sample is in a liquid medium in the one or more wells.

29. The method of claim 28, wherein the method further comprises contacting the liquid medium with a sensor.

30. The method of any of claims 20 to 29, further comprising detecting an analyte in a liquid medium in contact with the biological sample.

31. The method of claim 30, wherein the analyte is one or more of O2, H+, and CO2.

32. The method of any of claims 20 to 31, wherein the metabolic measurement is oxygen consumption rate (OCR).

33. The method of any of claims 20 to 32, wherein the metabolic measurement is extracellular acidification rate (ECAR).

34. The method of any of claims 20 to 33, wherein further comprising forming a measurement volume by lowering a plunger into the well.20250068-01 / 027644-8511 35. The method of claim 34, wherein the plunger is lowered to contact the ring.

36. The method of claim 34, wherein the plunger does not contact the biological sample.

37. The method of any of claims 34 to 36, wherein the method comprises moving the plunger with a plunger stroke of from 0.05 inch to 0.4 inch, alternatively about 0.2 inch.

38. The method of any of claims 34 to 37, wherein the method comprises moving the plunger with a stroke speed of from 0.04 inch / second to 0.32 inch / second, alternatively about 0.16 inch / second39. The method of any of claims 34 to 38, wherein the measurement volume is from about 0.5% to about 50% of an original volume of the well.

40. The method of any of claims 34 to 39, wherein the screen is configured to allow thorough perfusion of liquid medium and oxygen in the reservoir.

41. The method of any of claims 34 to 40, wherein the screen is configured to allow full mixing of reagents injected into the well.

42. An analysis system comprising:the apparatus of any one of claims 1 to 19;20250068-01 / 027644-8511 a device for obtaining metabolic measurements.

43. The system of claim 42, wherein the device for obtaining metabolic measurements comprises one or more plungers configured to move vertically within the wells.

44. The system of claim 43, wherein the one or more plungers are affixed to a cartridge.

45. The system of claim 43, wherein one or more sensors are disposed on the one or more plungers for detecting an analyte in a liquid medium in contact with the sample.

46. The system of claim 45, wherein the analyte is one or more of O2, H+, and CO2.

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