Microtiter Plate Lids and Magnetic Adapters

Abstract

A microtiter plate assembly is disclosed that includes a base having a number of wells, a lid having a number of protrusions corresponding to the number of wells, a magnetic plate adapter including at least one magnet, and a force distribution plate including or coated with a magnetic or paramagnetic material. The magnetic plate adapter and the force distribution plate provide the necessary amount of force to reduce warping of large microtiter plate assemblies without increasing the footprint of the microtiter plate.

Description

[Technical field]

[0001] The present invention relates to a microtiter plate lid and a magnetic adapter.

[0002] [Related Applications] This application claims priority to U.S. Provisional Patent Application No. 63 / 328,901, filed April 8, 2022, which is incorporated by reference herein in its entirety. [Background technology]

[0003] Biomedical analysis and imaging is involved in many diagnostic and therapeutic procedures, including visualizing external and internal anatomical and physiological structures, features, and systems, and evaluating complex biological events in the body at the organ, tissue, cellular, and molecular levels. For example, biomedical imaging and analysis techniques are particularly well suited to assay devices. An assay is a qualitative and / or quantitative analysis of an unknown analyte. In one aspect, for example, assay devices can be used to perform an analysis of the type and concentration of analytes in a cell sample. These types of devices are well suited to analyze live cells and provide useful information about metabolic processes occurring within the cells. For example, these devices can provide real-time cellular analyte measurements that provide a transparent window into key functions that drive cellular processes such as signaling, proliferation, activation, toxicity, and biosynthesis. More specifically, these devices can generate metabolic phenotypes in a relatively short amount of time, describing the cellular microenvironment. Summary of the Invention [Problem to be solved by the invention]

[0004] It is desirable to perform these analyses using microtiter plates with 96 or more wells while maintaining high sensitivity, such as by using small sample volumes or very short distances between the sensors and the cells. However, existing solutions lack the structural stability required for use with standard microtiter plates and are therefore subject to warping, which can lead to errors in the assay measurements and a lack of sensitivity and consistency. This problem has also been observed to generally worsen over time.

[0005] Attempts have been made to stabilize large microtiter plates by adding weights or clamps to minimize movement and warping. However, these solutions are not compatible with existing plate readers and therefore fail to meet the specifications set forth in the Society for Biomolecular Screening (herein referred to as "SBS Compliance") standard for size and weight. In addition, attempts have been made to utilize magnets, but the proposed solutions have failed to provide sufficient force, did not meet the weight requirements for plate readers, or measurements were prevented due to the size of the magnets.

[0006] In addition, existing products lack the consistent measurement height required to maintain high sensitivity with small sample volumes. For example, to achieve high sensitivity, protrusions or stoppers on a microtiter plate lid must extend the same distance into each well, but must not vary significantly between other wells and protrusions located on the same microtiter plate. However, attempts to solve this problem have failed to provide a solution that provides such a low coefficient of variation for microtiter plates with a large number of wells (e.g., fail to provide edge or center support for microtiter plates with 96 or more wells).

[0007] Thus, although instruments have been proposed to limit warpage and improve the sensitivity and consistency of microtiter plates, there remains a need for a microtiter plate assembly that exhibits low warpage even at large array sizes. It would also be beneficial to provide a microtiter plate assembly having a defined distance between stoppers / protrusions with a low coefficient of variation. It would further be beneficial to provide an assembly that is compatible with existing microtiter plates and plate readers. [Means for solving the problem]

[0008] The present disclosure is generally directed to a microtiter plate assembly including a base, a lid, a magnetic plate adapter, and a force distributing plate. Further, the base includes a plurality of wells in at least one outer row and at least one inner row, and the lid includes a plurality of protrusions corresponding to the plurality of wells. Additionally, at least a portion of the force distribution plate, the magnetic plate adapter, or both the magnetic plate adapter and the force distribution plate are formed from or have a magnetic or paramagnetic material disposed thereon or attached thereto. Here, the at least one magnet provides sufficient force to reduce warping of the lid such that the assembly exhibits a percent warp of about 30% or less (about 30%, or less than about 30%), where the percent warp is defined as the signal change ratio of the average fluorescent signal of at least one outer row relative to the average fluorescent signal of at least one inner row (average fluorescent signal).

[0009] The present disclosure is also generally directed to a microtiter plate assembly including a base, a lid, a magnetic plate adapter, and a force distribution plate, the base including a plurality of wells spaced apart such that each well is adjacent to a second well, the lid including a plurality of projections spaced apart corresponding to an array of wells such that each projection is adjacent to a second well, and the magnetic plate adapter including at least one magnet. Additionally, at least a portion of the force distribution plate, the magnetic plate adapter, or both the magnetic plate adapter and the force distribution plate is formed from or has a magnetic or paramagnetic material disposed thereon, or has a magnetic material attached thereto. Furthermore, the plurality of projections extend from a proximal end adjacent the lid to a distal end, and a height gap is defined between the distal end of each projection and a well base of each well. Here, the variation rate (difference rate) of the height gap between the protrusion and well and the adjacent second protrusion and second well is about 10% or less (about 10%, or less than about 10%).

[0010] In one aspect, the force distribution plate, the magnetic plate adapter, or both the magnetic plate adapter and the force distribution plate provide at least about 4 kilograms of force (kgf) to the lid. Further, in one aspect, the assembly exhibits a camber rate of about 15% or less (about 15%, or less than about 15%). In another aspect, the at least one magnet is magnetized vertically.

[0011] In a further aspect, the longitudinal length of at least one of the magnetic plate adapter and the force distribution plate is equal to or less than the longitudinal length of the base. Additionally or alternatively, in one aspect, the base includes an apron configured to releasably accommodate at least one magnet. Further, in one aspect, at least one of the magnetic plate adapter and the force distribution plate is formed from aluminum, steel, combinations thereof, or alloys or derivatives thereof. In another aspect, the microtiter plate assembly is configured to be compatible with a plate reader and / or is Society for Biomolecular Screening (SBS) compliant. In one aspect, at least one of the magnetic plate adapter and the force distribution plate has a thickness of about 12 mm or less (about 12 mm or less). In a further aspect, at least one of the magnetic plate adapter and the force distribution plate has a weight of about 600 g or less (about 600 g, or less than about 600 g).

[0012] Additionally or alternatively, in one aspect, at least one of the magnetic plate adapter and the force distribution plate includes one or more apertures. In a further aspect, the assembly includes 96 wells and 96 corresponding protrusions. In yet another aspect, each protrusion is about 0.8 mm. 2 More than (approx. 0.8 mm 2 of, or about 0.8 mm 2 In one embodiment, the distal end of each protrusion is angled at an angle of about 5° or greater (about 5° or greater) relative to the plane of the lid. Further, in one embodiment, at least a portion of the assembly is formed from an acrylate polymer or at least a portion of the assembly is formed from an acrylate polymer that is resistant to a radial cross-sectional area of ​​about 15 cm at 23° C. and 0% relative humidity. 3 / m 2 / 24 hour oxygen transmission rate (oxygen permeability). In another aspect, at least one of the lid and the force distribution plate can include a plurality of spacers.

[0013] The present disclosure is also generally directed to a method of measuring oxygen consumption rate (oxygen consumption rate) utilizing an assembly of any one or more of the discussed embodiments, the method including placing a sample and an oxygen-sensitive phosphorescent probe into a well, contacting the sample with a corresponding protrusion, and measuring the oxygen consumption rate with a fluorescent plate reader.

[0014] In one embodiment, the sample has a seeding density of less than 70,000 cells and the measurement is performed in 45 minutes or less. In a further embodiment, the seeding density is conducted at or below confluence of the selected cell type. In yet another embodiment, the sample volume is less than or equal to 100 μL, preferably the sample volume is less than or equal to 70 μL.

[0015] Other features and aspects of the disclosure are discussed in more detail below.

[0016] A full enabling disclosure is set forth in the specification below in more detail, including with reference to the accompanying figures. [Brief description of the drawings]

[0017] [Figure 1A] FIG. 1 is an exploded view of one embodiment of a microtiter plate assembly according to the present disclosure. [Figure 1B] FIG. 1B is an assembly diagram showing the embodiment of FIG. 1A. [Figure 1C] FIG. 1C is a cross-sectional view showing the embodiment of FIG. [Figure 2A] FIG. 1 illustrates one embodiment of a microtiter plate assembly according to the present disclosure. [Figure 2B] FIG. 2B shows an embodiment of a microtiter plate lid according to FIG. 2A. [Figure 3A] FIG. 2 is a bottom view of another embodiment of a microtiter plate lid according to the present disclosure. [Figure 3B] FIG. 3B shows the microtiter plate lid of FIG. 3A along cross section BB. [Figure 3C] FIG. 3B is a side view of the microtiter plate lid of FIG. 3A. [Figure 3D] FIG. 3B is a top view of the microtiter plate lid of FIG. 3A. [Figure 3E] FIG. 3D is a cross-sectional view taken along line DD in FIG. 3D. [Figure 3F] FIG. 3B is a perspective view of the microtiter plate lid of FIG. 3A. [Figure 4A] FIG. 1 shows another embodiment of a microtiter plate lid according to the present disclosure. [Figure 4B] FIG. 4B shows a microtiter plate base that corresponds to the microtiter plate lid of FIG. 4A. [Figure 4C] FIG. 4B shows an embodiment of the microtiter plate lid of FIG. 4A releasably attached to the microtiter plate base of FIG. 4B. [Figure 4D] FIG. 1 illustrates one embodiment of a microtiter assembly according to the present disclosure. [Figure 5A] FIG. 1 illustrates a further embodiment of a microtiter plate assembly according to the present disclosure. [Figure 5B] FIG. 5B is an enlarged view of the distal end of the projection of FIG. 5A. [Figure 5C] FIG. 13 is a close-up view of a distal end of a protrusion according to the present disclosure. [Figure 6] 1 is a graph of warpage over time of an assembly according to the present disclosure, measured in relative fluorescence units according to Example 1. [Figure 7] 1 is a graph of percent warpage over 90 minutes according to Example 2. [Figure 8A] FIG. 1 shows the test method and data of Example 4. [Figure 8B]FIG. 1 shows the test method and data of Example 4. [Figure 8C] FIG. 1 shows the test method and data of Example 4. [Figure 8D] FIG. 1 shows the test method and data of Example 4. [Figure 9A] FIG. 2 is an exploded view of another embodiment of a microtiter plate assembly according to the present disclosure. [Figure 9B] FIG. 9B is a top view of the lid of the microtiter plate assembly of FIG. 9A. [Figure 9C] FIG. 9B is a top view of a force distribution plate that can be used with the microtiter plate assembly of FIG. 9A. [Figure 10] 9C is a graph showing a comparison of the coefficient of variation of oxygen depletion measurements in wells of lids of the microtiter plate assemblies of FIGS. 9A and 9B utilizing spacers having various thicknesses compared to lids without spacers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Repeat use of reference characters in the present specification and drawings is intended to represent same or analogous features or elements of the invention.

[0019] [Definitions and Test Methods] As used herein, warping or warp can be defined as the movement of pillars and / or protrusions in the y-direction. This results in an increased height gap and therefore a need to increase sample volume, as discussed in more detail below. For example, if warping occurs during an oxygen depletion assay, inconsistencies in sample volume occur across the microplate, and thus different levels of oxygen depletion are measured across multiple sample wells or chambers.

[0020] Furthermore, warping or warping percentage according to the present disclosure can be measured by providing a 96-well plate, dissolving a phosphorescent reagent (e.g., MitoXpress Xtra in one example) in the fluid samples contained in the wells, measuring the signal from the reagent, calculating the average fluorescent signal from each row of wells, and calculating the percent intensity of each row, where the percent intensity is expressed as a percentage of the average signal of the middle row, as discussed in more detail in Example 1 below. (For example, for a 96-well plate, there should be 8 rows of 12 wells, as generally shown in Figures 4A-4C, where rows 3-6 or C, D, E, and F as shown in Example 1 and Figures 4A-4C are the inner rows, and rows 1, 2, 7, and 8 or A, B, G, and H are the outer rows.) That is, as discussed in more detail below, this method assumes that an increase in fluorescent signal indicates an increase in sample volume when all other conditions (e.g., temperature, ambient oxygen) remain constant.

[0021] As used herein, "translucency" or "semitransparent" refers to the light transmittance or optical density of a material in the range between transparent and opaque. Generally speaking, a translucent material allows light to pass diffusely. A translucent material allows greater levels of electromagnetic radiation in the visible spectrum to pass through the material than an opaque or substantially opaque material, but allows lower levels of electromagnetic radiation in the visible spectrum to pass through the material than a transparent or substantially transparent material. A translucent material can provide protection from phototoxicity while still allowing detection of cells. As a result, the cells cannot be observed from above the plate.

[0022] The oxygen concentration can be determined or indirectly queried by placing an oxygen-sensitive photoluminescent probe and a fluid test sample into multiple wells in a microtiter plate and ascertaining the oxygen concentration in each well of the covered microtiter plate by exposing the oxygen-sensitive photoluminescent probe in each well to excitation radiation that passes through a protrusion extending into each well or through the well bottom to cause an excited oxygen-sensitive photoluminescent material, measuring the radiation emitted by the excited oxygen-sensitive photoluminescent material through the protrusion and through the well bottom, and converting the measured emission to a target analyte concentration based on known conversion algorithms or measuring the probe / sensor signal in an intensity or lifetime mode. In one aspect, a photoluminescent probe suitable for such measurements is the MitoXpress® Xtra available from Agilent Technologies. Instruments suitable for reading oxygen-sensitive photoluminescent probes in cell samples are known and available from several sources, including the CLARIOstar plate reader from BMG Labtech GmbH of Ortenberg, Germany, and the Synergy HTX from BioTek, an Agilent Technologies company, however, it will be appreciated that the photoluminescent material can also include an indicator dye incorporated into an oxygen-permeable polymeric matrix, and an electrochemical sensor can be used to determine oxygen consumption.

[0023] As used herein, Oxygen Consumption Rate (OCR), which may also be referred to as oxygen depletion rate, may be calculated by sensing the consumed metabolite (O2) from a medium sample, which may then be reported in the form of a rate (change in analyte over time), or may be calculated by measuring or indirectly assessing the analyte concentration at a preselected time point (end point). Conversely, oxygen production may be determined by measuring the increase in oxygen concentration, which results in an increase in oxygen over time.

[0024] The change in oxygen consumption can be determined in a sealed or non-sealed system. In one embodiment, the definition of OCR includes that oxygen consumption is not determined in a sealed system, e.g., a system that allows oxygen back diffusion or substantial oxygen back diffusion to the sample, or that oxygen consumption is oxygen depletion in the sample corrected for oxygen back diffusion to the sample, or that oxygen consumption is oxygen depletion uncorrected for oxygen back diffusion to the sample, or that oxygen consumption is determined in a sealed system, e.g., a system that does not allow oxygen back diffusion or substantial oxygen back diffusion to the sample, or that oxygen consumption is equal or substantially equal to oxygen depletion in the sample. Furthermore, in one embodiment, oxygen consumption is determined directly or indirectly, e.g., inferred from a measured oxygen gradient, e.g., by measuring oxygen in a test well or at a preselected time point.

[0025] As used herein, the Extracellular Acidification Rate (ECAR) can be determined using a baseline or initial value for the proton flux of a cell sample, for example, a value based on a measurement of the proton flux of the cell sample made before the formation of the reaction mixture, and by determining the rate of proton flux after the formation of the reaction mixture. For example, as used herein, the (ECAR) can be calculated by sensing the metabolite (H+) consumed or produced in the medium sample, which can then be reported in the form of a rate (change in analyte over time), or by measuring the analyte concentration at a preselected time point (end point). In one embodiment, the change in the ECAR can be determined in a sealed or unsealed system.

[0026] As used herein, the terms "about," "approximately," or "generally," when used to modify a value, indicate that the value may be increased or decreased by 10%, e.g., 7.5%, 5%, e.g., 4%, e.g., 3%, e.g., 2%, e.g., 1%, and still remain within the scope of the disclosed embodiments. Additionally, the term "substantially free of," when used to describe the amount of a substance in a material, is not limited to total or complete absence, but can correspond to the absence of any appreciable or detectable amount of the described substance in the material. Thus, for example, a material is "substantially free" of a substance when the amount of the substance in the material is less than the precision of an industry-accepted instrument or test for measuring the amount of the substance in the material. In certain exemplary embodiments, a material is said to be "substantially free" of a substance when the amount of the substance in the material is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% by weight of the material.

[0027] [Detailed Description] Those skilled in the art should understand that this discussion is a description of exemplary embodiments only, and is not intended to limit the broader aspects of the present disclosure.

[0028] Generally speaking, the present disclosure is directed to a microtiter plate assembly including a lid having multiple protrusions, a base having multiple reciprocating chambers (e.g., wells) corresponding to each of the protrusions, a force distribution plate, and a magnetic plate adapter. That is, the present disclosure has unexpectedly discovered that when an assembly according to the present disclosure having a unique orientation and unique force is used, an assembly is provided that is compatible with SBS and plate readers, and provides minimal warpage and height clearance even in large microtiter plate assemblies while maintaining an extremely low coefficient of variation. That is, the present disclosure has discovered that the unique combination of a force distribution plate having low weight and high stiffness and a magnetic adapter configured to provide a unique weight force, as discussed in more detail below, allows for an assembly to be provided that has significantly reduced warpage and a low coefficient of variation for the height clearance of the protrusions.

[0029] For example, in one aspect, at least one of the force distribution plate and the magnetic plate adapter is at least partially formed from a naturally magnetic or paramagnetic material, such as steel, iron, nickel, cobalt, aluminum, platinum, tungsten, magnesium, combinations thereof, or alloys and derivatives thereof, or is coated with a material that provides magnetic properties to the substrate while also exhibiting sufficient rigidity. In particular, at least one of the force distribution plate and the magnetic plate adapter is formed from a material that exhibits a low tendency to warp or flex. For example, in one embodiment, at least one of the force distribution plate and the magnetic plate adapter is formed from a material that exhibits a warpage rate of about 30% or less, such as about 25% or less, such as about 22.5% or less, such as about 20% or less, such as about 17.5% or less, such as about 15% or less, such as about 12.5% ​​or less, such as about 10% or less, or any range or value therebetween, such as about 1% to about 30%, such as about 5% to about 25%, such as about 7.5% to about 20%, compared to at least one of the inner rows, utilizing a Starret Granite Surface Plate (2720-0M) with an electronic digital display to measure the deflection point of the outer rows. However, while it has been discussed above that only one of the force distribution plate and the magnetic holder can be formed from such a material, it should be understood that both the force distribution plate and the magnetic plate adapter are formed from a material that is magnetic and has a high degree of stiffness.

[0030] Alternatively, at least one of the force distribution plate or magnetic holder is formed from a material that has a low tendency to warp or bend but is non-magnetic. In such an embodiment, the force distribution plate includes one or more magnets in locations that correspond to the magnets in the magnetic holder. For example, in one embodiment, a paramagnetic material can provide a low tendency to warp or bend, but does not provide sufficient force of weight. Thus, in one embodiment, at least one of the force distribution plate or magnetic holder is formed from a non-magnetic or paramagnetic material, such as aluminum, and includes one or more magnets having a size and force, as described below, that correspond to the magnets on the magnetic holder. Additional benefits of utilizing a non-magnetic or paramagnetic material with one or more magnets can also be provided, such as heat treating the non-magnetic or paramagnetic material to increase the flatness of the force distribution plate before assembly. Thus, as used herein, "magnetic properties" can refer to properties exhibited by a magnetic or paramagnetic material, or a non-magnetic or paramagnetic material that includes one or more magnets.

[0031] Nevertheless, in addition to having a high degree of stiffness, the magnetic plate adapter, force distribution plate, or combination thereof also includes at least one, such as at least two, such as at least three, such as at least four, such as eight or less, such as seven or less, such as six or less magnets attached to the magnetic plate adapter. In other words, the magnetic plate adapter, force distribution plate, or combination thereof includes a magnet volume (or magnetic properties) sufficient to provide the assembly with a force of about 3 kilograms force (kgf) or more, such as about 3.5 kgf or more, such as about 4 kgf or more, such as about 4.5 kgf or more, such as about 5 kgf or more, such as about 6 kgf or more, such as about 7 kgf or more, such as about 8 kgf or more, such as about 9 kgf or more, such as about 10 kgf or more, such as about 20 kgf or less, such as about 19 kgf or less, such as about 18 kgf or less, such as about 17 kgf or less, such as about 16 kgf or less, such as about 15 kgf or less, such as about 14 kgf or less, or any range therebetween. In particular, the present disclosure has found that when one or more magnets are used in accordance with the force ranges described above, the assembly is capable of providing sufficient force to prevent warping and a magnetic field low enough to minimize or eliminate interference with measurements by one or more plate readers.

[0032] In addition, as noted above, the present disclosure has found that such force distribution plates and magnetic plate adapters can be sized to fit SBS compliance and existing plate readers without increasing the lateral or longitudinal (vertical) size of the microtiter plate adapter, but are also sized to have lateral and longitudinal dimensions closely similar to the microtiter plate holder to provide stabilization at the corners and center of the assembly.

[0033] For example, in one aspect, the longitudinal length of at least one of the force distribution plate and the magnetic plate adapter is about 99.9% or less, such as about 98% or less, such as about 97% or less, such as about 96% or less, such as about 95% or less, such as about 90% or more, such as about 91% or more, such as about 92% or more, such as about 93% or more, such as about 94% or more, or any range or value therebetween, however, it should be understood that in one aspect, the longitudinal length of at least one of the force distribution plate and the magnetic plate adapter is generally equal to the longitudinal length of the lid and / or base.

[0034] Similarly, in one embodiment, the lateral width of at least one of the force distribution plate and the magnetic plate adapter is about 99.9% or less, such as about 98% or less, such as about 97% or less, such as about 96% or less, such as about 95% or less, such as about 90% or more, such as about 91% or more, such as about 92% or more, such as about 93% or more, such as about 94% or more, or any range or value therebetween, of the lateral width of the lid and / or base. However, it should be understood that in one embodiment, the lateral width of at least one of the force distribution plate and the magnetic plate adapter is generally equal to the lateral width of the lid and / or base. For example, in one embodiment, the lateral width of at least one of the force distribution plate and the magnetic plate adapter is about 90% to about 99.9%, such as about 92% to about 98%, such as about 94% to about 97% of the lateral width of the lid and / or base, or any range or value therebetween.

[0035] In particular, the present disclosure has found that by utilizing a vertically magnetized magnet mounted generally perpendicular to the magnetic plate adapter, as discussed in more detail below, the magnet can be contained within the skirt of the base and still provide sufficient magnetic force through the base and lid to the force distribution plate without increasing the footprint of the assembly. Thus, the assembly of the present disclosure does not require an increase in the lateral width or longitudinal length of the assembly beyond a standard size microtiter plate assembly. For example, when using a 96-well microtiter plate assembly, the total footprint of an assembly according to the present disclosure can have a length of about 128 mm or less, a width of about 86 mm or less, and a height of about 20 mm or less, such as from about 125 mm to about 135 mm, such as from about 127 mm to about 130 mm, a width of about 80 mm to about 90 mm, such as from about 84 mm to about 88 mm, and a height of about 15 mm to about 25 mm, such as from about 19 mm to about 23 mm, or any range or value therebetween.

[0036] Further, in one embodiment, to allow the assembly to fit existing plate readers and SBS compliance, at least one of the force distribution plate and magnetic plate adapter has a height of about 10 mm or less, such as about 9 mm or less, such as about 7 mm or less, such as about 5 mm or less, such as about 4 mm or less, such as about 3.5 mm or less, such as about 3 mm or less, such as about 2.5 mm or less, such as about 2 mm or less, or any range or value therebetween. For example, in one embodiment, at least one of the force distribution plate and magnetic plate adapter has a height of about 1 mm to about 10 mm, such as about 1.5 mm to about 7.5 mm, such as about 1.75 mm to about 4.25 mm, or any range or value therebetween.

[0037] In one embodiment, both the force distribution plate and the magnetic plate adapter have a height according to the ranges above, or alternatively, both the force distribution plate and the magnetic plate adapter have a combined height of about 12 mm or less, such as about 11 mm or less, such as about 10 mm or less, such as about 8 mm or less, such as about 6 mm or less, such as about 5.5 mm or less, such as about 5 mm or less, such as about 4.5 mm or less, such as about 4 mm or less, or any range or value therebetween. For example, in one embodiment, the force distribution plate and the magnetic plate adapter have a combined height of about 3.5 mm to about 12 mm, such as about 3.75 mm to about 11 mm, such as about 3.75 mm to about 4.25 mm, or any range or value therebetween.

[0038] In addition, the present disclosure has found that the clamping type function provided by the magnetic force discussed above can provide sufficient force alone. Thus, in one embodiment, at least one of the force distribution plate and the magnetic plate adapter has a weight of about 600g or less, such as about 550g or less, such as about 500g or less, such as about 450g or less, such as about 400g or less, such as about 350g or less, such as about 300g or less, such as about 250g or less, such as about 200g or less, or any range or value therebetween. For example, in one embodiment, at least one of the force distribution plate and the magnetic plate adapter has a weight of about 150g to about 650g, such as about 175g to about 625g, such as about 190g to about 610g, or any range or value therebetween.

[0039] Further, in one embodiment, both the force distribution plate and the magnetic plate adapter have a combined weight of about 800g or less, such as about 750g or less, such as about 700g or less, such as about 650g or less, such as about 600g or less, such as about 550g or less, such as about 500g or less, such as about 450g or less, such as about 400g or less, or any range or value therebetween. For example, in one embodiment, the force distribution plate and the magnetic plate adapter have a combined weight of about 350g to about 850g, such as about 375g to about 750g, such as about 390g to about 500g or less, or any range or value therebetween.

[0040] In one aspect, the force distribution plate and / or magnetic plate adapter can be formed from a material that naturally has the weight described above due to its size and / or thickness. However, in one aspect, the present disclosure has found that by using a material with a high degree of stiffness, multiple openings can be formed in at least one of the force distribution plate and the magnetic plate adapter without compromising the force and stiffness characteristics described above. Thus, in one aspect, at least one of the force distribution plate and the magnetic plate adapter has multiple openings that penetrate the thickness of each of the force distribution plate and the magnetic plate adapter. In particular, in one aspect, the multiple openings can be spaced and / or sized to allow viewing of each well from the top surface down (top-down) or the bottom surface up (bottom-up). In such an aspect, each opening can correspond one-to-one with each well, or can provide a window through which multiple wells can be viewed. For example, in one aspect, the window opening can provide a view of about 1 or more wells, such as about 2 or more wells, such as about 4 or more wells. Additionally, in one aspect, the force distribution plate can have apertures shaped and sized to provide a field of view for each well individually, while the magnetic plate adapter is provided with window apertures. Nevertheless, it should be apparent to one of skill in the art that the apertures can be selected to provide a selected weight, stiffness, and field of view for the samples to allow the assembly to be used with existing plate readers.

[0041] Furthermore, as discussed above, the present disclosure has found that by utilizing an assembly according to the present disclosure, the projections or stops described herein exhibit a very precise height gap (the distance between the most distal point of the distal end and the underside of the chamber or well), thereby allowing improved sensitivity for the measurement of any measurable analyte that is dependent on sample volume, such as, by way of example only, oxygen consumption rate or extracellular acidification, without impeding cell growth, and in one aspect exhibiting improved consistency from well to well, row to row, column to column, or combinations thereof. In particular, when the stop tabs of the lid are in contact with the stop tab receivers of the base containing the multiple chambers, the average height gap between the most distal point of each projection of a microtiter assembly according to the present disclosure and the underside of a chamber or well can be about 0.25 mm or less, such as about 0.20 mm or less, such as about 0.15 mm or less, such as about 0.1 mm or less, such as about 0.075 mm or less, such as about 0.5 mm or less, or any range or value therebetween. For example, in one embodiment, the average height gap of the microtiter assembly is from about 0.025 mm to about 0.25 mm, such as from about 0.035 mm to about 0.15 mm, such as from about 0.045 mm to about 0.1 mm, or any range or value therebetween.

[0042] Furthermore, because the assembly including the lid and protrusions described above allows for a very consistent height gap, in one aspect the height gap can have a very low degree of variability, such that the variation (difference) in height gap between adjacent protrusions / wells is about 10% or less, such as about 5% or less, such as about 2.5% or less, such as about 2% or less, such as about 1% or less. In particular, the present disclosure has found that when a microtiter assembly is formed as described above, increased well-to-well, row-to-row, column-to-column, or combinations thereof sensitivity is realized even with reduced sample sizes because the force distribution plate and magnetic plate adapter provide a uniform force throughout the assembly, allowing each stop tab to properly contact the base and fully seat the protrusions into each respective well.

[0043] Nevertheless, as discussed above, assemblies according to the present disclosure also provide assemblies with reduced warpage, as discussed in more detail in the examples below. In particular, assemblies according to the present disclosure can exhibit warpage in an amount of about 25% or less, such as about 20% or less, such as about 15% or less, such as about 10% or less, as discussed above in relation to warpage percentage, and as discussed in more detail, flatness or warpage measurements were taken during stress testing, where the part was cycled between room temperature and 37°C. In addition, the above warpage values ​​can be exhibited when tested for about 30 minutes or more, such as about 45 minutes or more, such as about 60 minutes or more, such as about 75 minutes or more, such as about 90 minutes or more, such as about 105 minutes or more, such as up to about 120 minutes.

[0044] As will be apparent to one of skill in the art, the assemblies of the present disclosure can provide benefits to any microtiter well and lid combination with consistent sample size (e.g., consistent distance between the sensor and the sample or bottom of the well) and any measurable analyte. For example, exemplary microtiter devices that can benefit from use in assemblies according to the present disclosure can be generally illustrated by U.S. Patent Application Publication Nos. 2018 / 0292393, 2020 / 0166529, and 2014 / 0248650, which are incorporated herein in their entirety. Of course, it should be recognized that other microtiter devices can also benefit from use in assemblies according to the present disclosure, as discussed.

[0045] Additionally, in one aspect, microtiter wells and lids that may benefit from use in the assemblies of the present disclosure may include a lid having a plurality of protrusions extending longitudinally from the lid, each protrusion having a combination of a radial notch (indentation) and a beveled distal end, and the radial notches and beveled distal end may be formed from or coated with a material having a low oxygen transmission rate, allowing for reduced sample size while reducing ambient oxygen.

[0046] In particular, in one aspect, the present disclosure has discovered that by utilizing a specially sized radial notch extending lengthwise from the distal end to the proximal end of each protruding portion, air bubbles present within the sample chamber (e.g., well) can be evacuated while also preventing sample overflow. In one aspect, the radial notch is approximately 0.575 mm in diameter, as will be discussed in more detail in connection with the following figures. 2 More than this, for example, about 0.585 mm 2 More than this, for example, about 0.595 mm 2 More than, for example, about 0.6 mm 2 More than 0.65 mm, for example 2 More than, for example, about 0.7 mm 2 More than 0.75 mm, for example 2 More than 0.85 mm, for example 2 More than 0.95 mm, for example 2 More than 1.00 mm, for example 2 More than 1.1 mm, for example 2 More than 1.2 mm, for example 2 More than 1.3 mm, for example 2 More than 1.4 mm, for example 2 More than, for example, about 1.5 mm 2 More than 1.6 mm, for example 2 More than 1.7 mm, for example 2 More than 1.75 mm, for example 2 More than 1.8 mm, for example 2 More than 1.9 mm, for example 2 More than 2mm, for example 2 Or about 2 mm 2 For example, in one embodiment, the radial notch has a radial cross-sectional area of ​​less than about 0.85 mm 2 ~about 2.5mm 2 , for example, about 1 mm 2 ~about 2.25mm 2 , or about 1.25 mm 2 ~ approx. 2 mm 2, or any range or value therebetween. As discussed above, the present disclosure has surprisingly found that when the radial notch has a radial cross-sectional area sized according to the above, the radial notch allows gas bubbles to escape from the sample without contributing to reoxygenation.

[0047] In other words, in one aspect, the cross-sectional area of ​​the radial notch can be proportional to the cross-sectional area of ​​the proximal end of the protrusion. For example, the cross-sectional area defined by the proximal end of the protrusion can include the cross-sectional area defined by the radial notch. Thus, the radial cross-sectional area of ​​the radial notch can occupy about 5% or more, such as about 8% or more, such as about 10% or more, such as about 15% or more, such as about 20% or more, such as about 25% or more, up to about 30% or less than about 30%, such as about 5% to about 30%, such as about 8% to about 25%, or such as about 10% to about 20%, or any range or value therebetween. As discussed above, the present disclosure has surprisingly found that when the radial notch has a radial cross-sectional area sized according to the above, the radial notch can allow air bubbles to escape from the sample without contributing to reoxygenation or overflow. As discussed above, any diameter protrusion can be used relative to the notch, however, in one embodiment, at least one protrusion has a diameter of from about 1 mm to about 5 mm, e.g., from about 1.5 mm to about 4 mm, e.g., from about 2 mm to about 3 mm, or any range or value therebetween.

[0048] Further, the radial notches can have any cross-sectional shape, such as circular, oval, rectangular such as square, triangular, nodular, etc. However, in one aspect, the radial notches can have a circular cross-section, e.g., circular or elliptical cross-section. Thus, in such an aspect, as will be discussed in more detail in connection with the following figures, the radial notches extend in the sidewall of each protrusion from the distal end to the proximal end throughout the length of the respective protrusion without penetrating or piercing the sidewall of the protrusion. In other words, the radial notches form a portion of the sidewall of each protrusion and do not extend into the interior of the protrusion. Thus, it should be clear that the notches or channels formed by the radial notches are formed between the portion of the sidewall formed by the radial notch and the sidewall of a chamber or well that conforms (matches) with the respective protrusion, and are not formed in the interior (e.g., hollow) of the protrusion.

[0049] In addition, by using radial notches having the above sizes and dimensions, the radial notches allow excess sample to be contained between the respective protrusion body and the sidewall of the sample chamber or well without spilling over the chamber or well and contaminating an adjacent chamber or well. Thus, in such an embodiment, the distal end of the protrusion is at least partially contacted by or fully immersed in the sample, thus containing some sample within the channel formed between the radial notch and the respective conformal chamber or well. However, it is noted that in one embodiment, each protrusion has only one notch (whether for oxygen dissipation or overflow for other applications). That is, when shaped and sized according to the above, only a single radial notch is needed to provide an area for dissipating air bubbles and containing overflow. However, in further embodiments, one or more of the protrusions can include two notches, three notches, or four notches. In such an embodiment, each notch can be shaped and sized as discussed herein. As discussed in more detail below, in one embodiment, at least one post has two notches formed symmetrically about a centerline extending from the proximal end to the distal end of the post.

[0050] In one such embodiment in which the protrusions include at least two notches, the distal ends of the protrusions can include a spotting surface that is generally parallel to the plane of the lower surface or lid of the respective well. For example, the spotting surface can have generally the same cross-sectional shape as the respective protrusions and can have a diameter of about 0.5 mm to about 4.5 mm, e.g., about 1 mm to about 4 mm, e.g., about 1.5 mm to about 3.5 mm, or any value or range therebetween. Further, in one embodiment in which one or more of the lid, protrusion, base, or well are formed to have an opaque color, the spotting surface can be generally clear or matte.

[0051] However, in one aspect, the sample may completely surround or contact the distal ends of at least some or all of the protrusions, but based on the description of the method of determining the oxygen consumption rate, it should also be understood that the oxygen consumption rate is not determined based on visual inspection from above the sample. Instead, the oxygen consumption rate is determined in one aspect utilizing a signal detector, such as a plate reader, that measures the photoluminescent material, as discussed in more detail below. Thus, in one aspect, the substrate material used to form some or all of the protrusions, the cover lid, or the base including the chambers or wells corresponding to the protrusions, is formed from a translucent material, which is distinguished from a transparent material by limiting the view of the sample through the material while still allowing enough light to pass through to enable interrogation of the probe. In particular, such a material may allow the photoluminescent material to be measured by the above method from a top-down plate reader, a bottom-up plate reader, or a combination thereof, while diffusing the light, as known in the art. In one aspect, at least one of the base, the lid, and the one or more protrusions can be translucent, can have a black, white, or matte appearance, or can be transparent.

[0052] Additionally, the distal end of each longitudinal projection (e.g., the end of each projection opposite the end adjacent the lid) can be inclined or sloped at a particular angle with respect to a plane parallel to the lid (or well bottom) such that the highest side of the distal end (e.g., the side of the distal end that has a shorter length from the distal end to the lid) is adjacent to a radial notch, which guides any air bubbles present in the sample into the notch and facilitates (facilitates / assists) their removal. Thus, in one embodiment, the distal end is inclined or sloped at an angle of about 5° or more, such as about 7.5° or more, such as about 10° or more, such as about 12.5° or more, such as about 15° or more, such as about 20° or more, such as about 25° or more, up to about 30° or less, with respect to the lid or well bottom. For example, in one embodiment, the distal end is beveled or sloped at an angle of about 3° to about 30°, e.g., about 3° to about 25°, e.g., about 5° to about 20°, or any range or value therebetween. In particular, when angles according to the above are used with the distal end sloped towards the radial notch, air bubbles can be substantially removed from the sample and can also allow for very sensitive oxygen consumption rate readings with small sample sizes.

[0053] It will further be appreciated that in embodiments including one or more protrusions with two or more notches, the protrusions may also include two or more angled portions (sloped portions) at the distal tip corresponding to the two or more protrusions. For example, in an embodiment including two protrusions, the sloped distal tip may include two angled portions, each having a highest side adjacent a respective radial notch and having a distal-most point that intersects approximately at the longitudinal centerline. In this manner, air bubbles may be substantially removed into the one or more notches for removal from the sample.

[0054] For example, lids according to the present disclosure can be used to measure oxygen consumption rates, among other measurements, and can utilize fill volumes of 100 μL or less, such as about 90 μL or less, such as about 80 μL or less, such as about 70 μL or less, such as about 60 μL or less, such as about 60 μL or less, such as about 50 μL or less, such as about 40 μL or less, such as about 30 μL or less, such as about 25 μL or less, such as about 20 μL or less, such as about 15 μL or less, such as about 10 μL or more, such as about 15 μL or more, or any range or value therebetween. For example, in one embodiment, the fill volume can be from about 10 μL to about 100 μL, such as from about 15 μL to about 90 μL, such as from about 20 μL to about 80 μL, or any range or value therebetween.

[0055] In other words, the plate lid according to the present disclosure allows lower seeding density to be used while maintaining measurable results.For example, in one aspect, the plate lid according to the present disclosure can be utilized with seeding density of about 100,000 cells or less, such as about 75,000 cells or less, such as about 50,000 cells or less, such as about 25,000 cells or less, such as about 12,500 cells or less, such as about 10,000 cells or less, such as about 7,500 cells or less, such as about 5,000 cells or less, such as about 2,500 cells or less, such as about 1,000 cells or more, such as about 2,500 cells or more, or any value or range therebetween. For example, in one embodiment, the seeding density is from about 2,500 cells to about 125,000 cells, such as from about 6,500 cells to about 90,000 cells, such as from about 10,000 cells to about 75,000 cells, or any range or value therebetween.

[0056] That is, as described above, by utilizing the assembly according to the present disclosure, increased sensitivity and improved measurement consistency can be achieved. Thus, the assembly according to the present disclosure can provide benefits to fluid samples containing various cell lines, for example, by increased sensitivity and reduced oxygen ingress / back diffusion. That is, as discussed above, the assembly according to the present disclosure can be used with adherent cell lines known in the art in addition to suspension cell lines. As understood in the art, suspension cell lines can be immobilized on coated plates (e.g., by using a centrifuge and immobilization coatings such as PDL, PLL, collagen, and fibronectin, as discussed in more detail below) and analyzed using any one or more of the methods described herein.

[0057] Additionally, the assemblies of the present disclosure can also be utilized with and provide benefits to 3D structures, examples of which include spheroids immobilized on coated plates (examples of which are described in U.S. Provisional Patent Application No. 63 / 276,099, which is incorporated herein in its entirety) and / or organoids, 3D culture systems utilizing scaffolds or matrix materials (e.g., electrospun fibrous scaffolds, collagen, hydrogels, and Matrigel, including Lonza's RAFT™ 3D cell culture system), tissues and microtissues, and magnetic spheroids.

[0058] For example, one such magnetic spheroid method that can benefit from small sample sizes includes magnetic spheroid drive. As known in the art, magnetic spheroids can be measured or localized using a variety of methods, for example in combination with low adhesion plates, magnetic particles, and magnets (e.g., using polylysine, magnetic arrays, etc.). One example of a spheroid workflow includes, by way of example only, the Greiner workflow, where cells are magnetized and formed into spheroids utilizing magnetic drive. After spheroid formation, the spheroids can be maintained within the Greiner spheroid plate or moved to another suitable microplate. Regardless of the plate selected, the spheroids can be localized using magnetic drive while inserting a lid according to the present disclosure to orient the spheroids at the base of the well. Furthermore, in one embodiment, the magnetic drive can be maintained during the assay to localize the spheroids.

[0059] Additionally or alternatively, one or more of the protrusions can be hollow, as described above. Thus, in one aspect, the hollow core of one or more of the protrusions can include a core magnet to aid in localizing the spheroids, allowing the lid to be deployed without magnetic actuation. In one aspect, one or more of the protrusions include a core magnet permanently attached to the respective hollow. Conversely, in one aspect, a lid assembly according to the present disclosure can include a protrusion overlay disposed between the protrusions and the cover block. The protrusion overlay includes one or more magnetic spines releasably extending in the hollow of one or more of the protrusions, such that such magnetic spines can be removed after localizing the spheroids or transporting the spheroids. In one such aspect, the protrusion overlay can include a number of magnetic spines corresponding to the number of protrusions, such that when the protrusion overlay is deployed, the hollow of each protrusion is at least partially occupied by a magnetic spine.

[0060] Moreover, it should be understood that other fluid samples can benefit from a lid according to the present disclosure, as will be appreciated by those skilled in the art. For example, the present disclosure can be used with enzymes, isolated organelles such as mitochondria, microorganisms, prokaryotes, eukaryotes, stem cells, cardiomyocytes, ex vivo samples embedded in microscope slides, etc. Nevertheless, while examples of various cell lines and samples have been provided, it should be understood that in accordance with the present disclosure, in one aspect, any sample suitable for interrogation using a chamber or well according to the present disclosure can be used that benefits from tailored sensitivity, reduced or controlled oxygen ingress / back diffusion, reduced sample volume (such as increased cell to volume ratio), or combinations thereof.

[0061] Furthermore, as will be appreciated by those skilled in the art, one or more culture coatings can be used on the plate, protrusions, lid, or combinations thereof based on the fluid sample selected. For example, as discussed above, coatings for suspension cell lines can include poly-D-lysine (PDL), poly-L-lysine (PLL), collagen, and fibronectin. Furthermore, for spheroid assays, ultra-low attachment coatings, other common tissue culture coatings, or combinations thereof can be used. For example, suitable ultra-low attachment coatings include BioFloat® Flex available from faCellitate and Lipidure® available from NOF Corporation, which help to avoid ambiguous surface binding of proteins and cells. However, it should be apparent that other culture coatings can also be used as known in the art.

[0062] Nonetheless, in one aspect, a lid according to the present disclosure may reversibly contact the base and thus not be permanently attached to the base. That is, each projection may be reversibly associated with a respective chamber or well. For example, in one aspect, a stop tab on the lid may maintain contact with the base for a desired testing period using the plate assemblies described herein.

[0063] To achieve reversible attachment, the present disclosure has found that a specific wall gap, defined as the distance between any portion of the sidewall of each protrusion and the sidewall of the chamber or well, can be used to allow the lid to be removed when excess sample is returned to the chamber or well. That is, if too small a wall gap is used, the lid will not be removable, but if the wall gap is too large, air bubbles will be trapped between the portions of the sidewall that do not include the radial notch, reducing the ability to fully evacuate the air bubbles. Thus, in one aspect, an assembly according to the present disclosure has a wall gap of about 0.5 mm or less, such as about 0.4 mm or less, such as about 0.3 mm or less, such as about 0.2 mm or less, such as about 0.175 mm or less, such as about 0.15 mm or less, or any range or value therebetween. For example, in one embodiment, the wall gap is from 0.025 mm to about 0.5 mm, such as from about 0.05 mm to about 0.4 mm, such as from about 0.75 mm to about 0.3 mm, or any range or value therebetween.

[0064] In other words, the wall gap should be significantly smaller than the gap caused by the radial notch in order to allow air bubbles to escape through the notch rather than trapping the air bubbles between the sidewall of the protrusion and the sidewall of the corresponding well. Thus, in one aspect, the radial notch defines a radial notch gap defined as the distance between a point having a maximum distance from the sidewall of the respective well along a radial cross section (e.g., the apex of the radial notch) and the respective well sidewall, and the wall gap is about 50% or less of the radial notch gap, such as about 40% or less of the radial notch gap, such as about 30% or less, such as about 20% or less, such as about 10% or less, such as about 5% or less, such as about 2.5% or less, such as about 1% or less.

[0065] Furthermore, since the lid assembly, including the lid and the protrusions described above, allows for a very consistent wall gap, in one embodiment, the wall gap can have a very low degree of variability, such that the wall gap variation between adjacent protrusions / wells is about 10% or less, such as about 5% or less, such as about 2.5% or less, such as about 2% or less, such as about 1% or less. In particular, the present disclosure has found that when a microtiter assembly is formed as described above, increased sensitivity is achieved even at reduced sample sizes. However, it should be understood that in one embodiment, such as an embodiment in which the assembly is not intended to be reused, the lid can be permanently attached to the base after it has been placed in relation to the base.

[0066] Regardless of the method used, a further advantage of the disclosed assemblies is that evaporation is limited due to small wall gaps while maintaining reversibility. Thus, unlike samples that utilize fixed or permanent lids, as well as oil-in headspace, samples according to the present disclosure can undergo further testing after cellular respiration measurements are completed. For example, the disclosed assemblies should allow for protein assays or fluorescence-based assays to be completed after oxygen consumption rate testing, such as post-assay immunofluorescence assays (including, for example, TOM20 and LC-3).

[0067] Nevertheless, it should also be understood that in addition to the subsequent tests, additional simultaneous tests can be performed with the fluorescence-based assay, which can also be referred to as "multiplexing." That is, as discussed in more detail, the assembly of the present disclosure is SBS (Society for Biomolecular Screening) compatible and thus compatible with existing signal detectors for measuring additional target analytes (in addition to H+, CO, CO2, O2, discussed in more detail herein), including calcium, ATP, NADP / NADHP, temperature, mitochondrial membrane potential, reactive oxygen species, etc. Thus, it should be understood by those skilled in the art that any additional test that does not interfere with the target analyte can be performed simultaneously and multiplexed, but in one aspect, the additional test can include dyes (such as Hoechst and Calcein AM), fluorescent dyes, mitochondrial membrane potential (JC-1, e.g., TMRE / TMRM), reactive oxygen species (DHE), or combinations thereof.

[0068] Alternatively, multiplexed analytes may require the use of separate signal detectors that do not interfere with the target analytes discussed herein. The additional signal detectors may have one or more detection modes, such as detection of absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, fluorescence polarization, imaging, including fluorescence imaging, or combinations thereof. Plate readers with these detection modes are commercially available. Alternatively, in one aspect, the sensor may be interrogated from below, such as from below the lid of the device, and in such an arrangement, the signal should be able to pass through the wells and / or chambers so that the signal can be detected, for example by the wells and / or chambers including transparent or semi-transparent materials, or by allowing interrogation from above without requiring transparent materials at the distal end of the protrusions, for example by having one or more optical fibers passing through or contained within the lid and / or protrusions.

[0069] In addition, as briefly described above, and as will be appreciated by those skilled in the art, the substrate material forming the base, lid, protrusion, or combination thereof has increased contact with the sample due to its ability to fill the chamber, as well as the absence of an oil barrier between the sample and the headspace or lid. Thus, as will be apparent to those skilled in the art, the assembly of the present disclosure allows for the selection of sample and / or fill volume, substrate material, and optional low-oxygen permeable coating based on the cell line to be studied. That is, it is well known that different cells respire differently and therefore deoxygenate samples at different rates. For example, in one aspect, cells that respire at a higher rate may require a larger fill volume or may have a lower sensitivity to substrate materials or coatings, while less active cell lines may benefit from smaller sample volumes and coatings or substrate materials that limit the back diffusion of oxygen. Nevertheless, in one aspect, the material used to form or coat the substrate may have a minimum sample volume of about 600 cm when measured at 23° C. and 0% RH according to ASTM D3985. 3 / m 2 / 24 hours or less, for example, about 300 cm 3 / m 2 / 24 hours or less, for example, about 150 cm 3 / m 2 / 24 hours or less, for example, about 120 cm 3 / m 2 / 24 hours or less, for example, about 100 cm 3 / m 2 / 24 hours or less, for example, about 80 cm 3 / m 2 / 24 hours or less, for example, about 50 cm 3 / m 2 / 24 hours or less, for example, about 25 cm 3 / m 2 / 24 hours or less, for example, about 16 cm 3 / m 2 / 24 hours or less, minimum approximately 0cm 3 / m 2 / 24 hours or approx. 0cm 3 / m 2 / Has an oxygen transmission rate above or below 24 hours.

[0070] In addition, in one embodiment, when low oxygen back diffusion is required for sensitive samples, the material used to form or coat the substrate has a back diffusion coefficient of less than about 16 cm when measured at 23° C. and 0% RH according to ASTM D3985, as discussed in more detail in the Examples below. 3 / m 2 / 24 hours or less, for example, about 15 cm 3 / m 2 / 24 hours or less, for example, about 12.5 cm 3 / m 2 / 24 hours or less, for example, about 10 cm 3 / m 2 / 24 hours or less, for example, about 7.5 cm 3 / m 2 / 24 hours or less, for example, about 5 cm 3 / m 2 / 24 hours or less, for example, about 2.5 cm 3 / m 2 / 24 hours or less, for example, about 1 cm 3 / m 2 / 24 hours or less, for example, about 0.5 cm 3 / m 2 / 24 hours or less, or any range or value therebetween. For example, in one embodiment, the material has a low oxygen transmission rate of about 0.01 cm 3 / m 2 / 24 hours ~ approx. 20cm 3 / m 2 / 24 hours, for example, about 0.05 cm 3 / m 2 / 24 hours ~ approx. 17.5cm 3 / m 2 / 24 hours, for example, about 0.1 cm 3 / m 2 / 24 hours - approx. 15cm 3 / m 2 / 24 hours, or any range or value therebetween. Such low oxygen back diffusion can assist in applications such as cancer research where the necessary cell lines require low cell densities, which is made possible by the use of the lids according to the present disclosure discussed herein.

[0071] For example, in one aspect, any portion of the assembly can be formed from matrix materials having a variety of morphologies and compositions, and can be derived from naturally occurring materials, synthetically modified naturally occurring materials, or synthetic materials. Examples of suitable matrix materials include, but are not limited to, nitrocellulose, glass, silica, Teflon, metals (e.g., gold, platinum, etc.), and ceramics (including aluminum oxide, silicon oxide, etc.), composites, and laminates thereof. Suitable matrix materials include polysaccharides such as agarose (commercially available, for example, from Pharmacia under the trade names Sephadex® and Sephacyl®, also from Pharmacia); polyacrylamides, polystyrenes, polyvinyl alcohols, copolymers of hydroxyethyl methacrylate and methyl methacrylate, polyesters including poly(ethylene terephthalate) and poly(butylene terephthalate), polyamides (such as nylons), polyethers including polyformaldehyde and poly(phenylene sulfide); polystyrenes available under the trade names KAPTON (DuPont, Wilmington, Del.) and UPILEX (Ube Industries, Inc.); Also included are polymeric materials including polyimides such as those manufactured by Polyolefin Polymers, Inc. (Kosan Co., Ltd., Japan), cyclic olefin polymers, ABS polymers, Kel-F copolymers, poly(methyl methacrylate), poly(styrene butadiene) copolymers, poly(tetrafluoroethylene), poly(ethylene vinyl acetate) copolymers, poly(N-vinylcarbazole), polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, polyolefin compounds including cycloolefin polymers (COP) sold under the trade names Zeonor® and Zeonex®, and cycloolefin copolymers (COC) sold under the trade name Topas®, and mixtures thereof. Specific polymeric materials that can be used for the substrate material include organic polymers that are naturally occurring or synthetic, crosslinked or uncrosslinked, homopolymers or copolymers.

[0072] For example, in one embodiment, the substrate material for all or a portion of the lid and base can be selected to be a material that has a naturally low oxygen permeability, such as an acrylate polymer, which can be polymethylmethacrylate, in one embodiment polyethyleneterephthalate, either alone or further coated with a polymer that has a very low oxygen permeability. In one embodiment, any of the above substrate materials can be used, but at least a portion of the substrate material is coated with any compound suitable for atomic layer deposition (ALD), molecular vapor deposition, or plasma-enhanced chemical vapor deposition (PECVD), such as a polymer or ceramic compound, for example a glass compound, or any compound suitable for low temperature deposition processes, such as Al2O3 or SiO2, ceramics and polymers, including silicon oxide, silicon nitride, silicon oxynitride, aluminum oxide, mixed oxides, flexible glass (alternating Al2O3 and polyacrylate layers on a PEN-based substrate, available as ClearProtect(C) 3D barrier coating from Antec). Other surface treatments are generally available, including Actiplas, Aquacer, Carbocer, Lipocer, Plasmaclean, Silitec, or combinations thereof. Additionally, in one embodiment, all of the sample contacting portions of the plurality of protrusions are coated with a material having very low oxygen permeability, either alone or in combination with coatings on other portions of the assembly. In particular, such coatings can further assist in preventing re-oxygenation of the sample after removal of the gas headspace.

[0073] Additionally, those skilled in the art will appreciate that additional reagents may be stored on the lid and / or protrusions by lyophilizing or solubilizing the reagents in a reagent matrix to further tailor the assay. Exemplary reagent substrates include D-glucose-6-PO4, pyruvate, α-ketoglutarate, α-ketobutyrate, Ala-Gin, Sparker Malate Control, α-D-glucose, citric acid, succinic acid, D,L-β-hydroxy-butyrate, L-serine, acetyl-L-carnitine, γ-amino-butyric acid, glycogen, D,L-α-glycerol-PO4, D,L-isocitrate, fumaric acid, L-glutamic acid, L-ornithine, octanoyl-L-carnitine, α-ketoisocaproic acid, D-glucose-1-PO4, L-lactic acid, cis-aconitic acid, L-malic acid, L-glutamine, tryptamine, palmitoyl-D,L-carnitine, L-leucine, and combinations thereof. Further, exemplary reagents may include metabolic regulators such as FCCP, rotenone, antimycin A, BAM15, and / or oligomycin, substrate catalysts, enzymes including glucose oxidase, and exemplary inhibitors include complex I inhibitor rotenone, complex II inhibitor malonate, complex III inhibitor antimycin A, uncoupler FCCP, ionophore K valinomycin, gossypol, polymyxin B, complex I inhibitor pyridaben, complex II inhibitor carboxin, complex III inhibitor myxothiazol, uncoupler 2,4-dinitrophenol, calcium, CaCl2, nordihydroguaiaretic acid, amitriptyline, meclizine, berberine, alexidine, phenformin, diclofenac, celastrol, trifluoperazine, papaverine, or combinations thereof. However, it should be understood that other reagent substrates, reagents, and inhibitors known in the art may also be used.

[0074] As discussed, in one aspect, the assembly according to the present disclosure is directed to a microtiter well plate assembly. Thus, the plate cover and base can have 6, 8, 24, 96, 384, 1536, etc. corresponding projections / wells, as known in the art. However, in one aspect, the assembly of the present disclosure is particularly well suited for microtiter plate assemblies having 96 or more wells and corresponding projections.

[0075] Exemplary aspects of the present disclosure are directed to assemblies and processes for analyzing one or more biological components, including cellular parameters, contained in or associated with a biological material sample, such as a cell culture. The processes and systems utilize optical detection and ranging components to not only efficiently perform readings, but also to perform measurements faster than many conventional systems. However, in one aspect, the probe and corresponding sensor are H + It should be understood that the assemblies may be selected to be any device capable of sensing and reporting changes in a target analyte, such as CO, CO2, O2, or combinations thereof. Furthermore, as discussed above, it should be understood that assemblies according to the present disclosure are suitable for identifying extracellular, intracellular, and pericellular analytes.

[0076] For example, oxygen-sensitive photoluminescent probes are widely known that are capable of sensing and reporting the oxygen concentration of the environment in fluid communication with the probe. See, for example, U.S. Patent Application Publication Nos. 2011 / 0136247, 2009 / 0029402, 2008 / 199360, 2008 / 190172, 2007 / 0042412, and 2004 / 0033575, U.S. Patent Nos. 8,242,162, 8,158,438, 7,862,770, 7,849,729, 7,749,768, 7,679,745, 7,674,626, 7,569,395, 7,534,615, 7,368,153, 7,138,270, 6 ,989,246, No. 6,689,438, No. 6,395,506, No. 6,379,969, No. 6,080,574, No. 5,885, No. 843, No. 5,863,460, No. 5,718,842, No. 5,595,708, No. 5,567,598, No. 5,462,879, No. See, for example, US Pat. Nos. 5,407,892, 5,114,676, 5,094,959, 5,030,420, 4,965,087, 4,810,655, and 4,476,870, WO 2008 / 146087, and European Patent Application Publication No. 1 134 583. Such optical sensors are available from a number of sources, including Presens Precision Sensing GmbH of Regensburg, Germany, Oxysense of Dallas, Texas, USA, and Agilent Technologies of Cork, Ireland.

[0077] Methods and techniques for sensing oxygen in test tubes or wells of microtiter plates using oxygen-sensitive photoluminescent probes are widely known, as exemplified by WO 2012 / 052068, U.S. Patent Application Publication No. 2013 / 0280751, and U.S. Patent Application Publication No. 2014 / 0147882, all of which are incorporated herein by reference, and are suitable for use in determining the oxygen concentration in a test tube or well sealed by an apparatus according to the present invention.

[0078] However, while discussed thus far using phosphorescent sensors, it should be understood that optical sensors according to the present disclosure may also utilize solid-state (semiconductor, solid state), nanoparticle, microparticle, and / or magnetic sensors, etc. For example, solid-state sensors may include one or more spots or films on a lid, base, protrusion, or combinations thereof, while particle-based sensors may generally be located in a solution or suspension. Alternatively, in one aspect, particle-based sensors may be loaded into a cell or coated on a surface. Nevertheless, such sensors may include light, O2, pH, temperature, CO2, or combinations thereof, as known in the art.

[0079] Further, in one aspect, the sensor can be an electrochemical or potentiometric sensor. Additionally or alternatively, electrodes can be included in the well to measure electrical properties including impedance. Regardless of the sensor selected, it should be understood that in one aspect, as discussed above, the well or chamber can also include one or more reference probes that generate a signal of known value for instrument calibration in any of the forms of sensors discussed above.

[0080] Regardless of the type of solid-state sensor selected, in one aspect, the sensor can be embedded in a permeable medium, for example, a permeable medium selected from hydrogel, silicone, and matrigel, by way of example only. In some aspects, the sensor is attached to at least one of the protrusions by solidifying or removing the medium (such as by drying, curing, cooling, evaporation, or other techniques). The solid-state sensor can be applied by dipping or spotting a distal end of at least one of the protrusions into a mixture of the fluorescent indicator in the medium.

[0081] However, it should be understood that in certain aspects, the sensor can be disposed or immersed into all or a portion of one or more of the protrusions. It should be further understood that in certain aspects, the sensor can be removably connectable to the body of one or more of the protrusions of the assembly. It should be further understood that in certain aspects, the sensor can be integrally formed with one or more of the protrusions. Integrating the sensor onto one or more of the protrusions can be accomplished by one or more techniques, such as vapor deposition, chemical coating, spin coating, immersion, and robotic spotting.

[0082] The assembly and process according to the exemplary aspects of the present disclosure can be well suited to measure components in all different types of samples, such as biological samples. In one aspect, for example, the system and process according to the exemplary aspects of the present disclosure can be used to measure one or more components or parameters related to components in cellular material. The one or more components can be contained in the medium surrounding the cell or can be contained within the cell itself. In some embodiments, the biological sample being tested can include cellular material derived from the cell, such as organelles, mitochondria, or cell extracts. It is particularly advantageous that these measurements can be completed without labels.

[0083] In one embodiment, the cell sample is obtained or derived from a subject, such as a human or non-human animal. In one embodiment, the subject is a mouse, and in one embodiment, has or is at risk of having a disorder. Nevertheless, in one embodiment, the cell sample can include primary cells, cells isolated or taken directly from a living tissue or organ, cultured cells, and / or immortalized cells. For example, the cell sample can include primary cells, or cells isolated or taken directly from a living tissue or organ and then cultured in vitro. In one embodiment, the cell sample includes modified cells, such as cells genetically engineered for heterologous expression of a gene of interest, and / or cells genetically engineered for inhibited expression of a gene, such as cells from a knockout mouse or a CRISPR KO library. Nevertheless, in one embodiment, the cell sample includes stem cells or cells derived from stem cells. Nevertheless, regardless of the cells used, in one embodiment, the cell sample includes a medium, such as a culture medium or growth medium, and the cells can be disposed within the medium. Moreover, as should be understood, in one aspect, a cell sample comprises a plurality of cells, such as a plurality of cells described herein.

[0084] The cells being tested can include any suitable cell sample, including, but not limited to, cultured cells, primary cells, human cells, neuronal cells, T cells, B cells, epithelial cells, muscle cells, stem cells, induced pluripotent stem cells, immortalized cells, pathogen-infected cells, bacterial cells, fungal cells, plant cells, archaeal cells, mammalian cells, avian cells, insect cells, reptilian cells, amphibian cells, and the like. The cells being tested also include three-dimensional cell samples, such as tissue samples, cell spheroids, biopsy samples, cytoskeleton, organ chips, and the like. Examples of parameters related to the above cell functions and that can be measured include carbon dioxide concentration, pH, oxygen concentration or oxygen tension, calcium ions, hydrogen ions, and the like. However, in one aspect, the parameter measured is oxygen concentration, such as oxygen depletion, or a related derivative measurement, such as oxygen consumption or cellular oxygenation. These tests can provide an understanding of what drives the phenotype and function of the cells, and / or an accurate picture of the cellular environment or microenvironment.

[0085] The assemblies and processes according to exemplary aspects of the present disclosure can be used to measure metabolic data of living cells or (micro)environmental conditions of any living cells. The cellular material being tested can include, for example, bacterial cells, fungal cells, yeast cells, prokaryotic cells, eukaryotic cells, etc. Cells that can be tested include mammalian cells, including animal cells and human cells. Particular cells that can be tested include cancer cells, immune cells, immortal cells, primary cells, induced pluripotent stem cells, cells infected with viral or bacterial pathogens, etc.

[0086] For example, in one aspect, the assemblies and processes according to exemplary aspects of the present disclosure can be used to support immunotherapy. Immunotherapy is a type of treatment that boosts a patient's immune system to fight cancer, infectious diseases, and other diseases. Immunotherapy processes can include, for example, adoptive cell therapy, such as the production of T cells, natural natural killer (NK) cells, monocytes, macrophages, combinations thereof, and the like. For example, during T cell therapy, T cells are removed from the patient's blood. The T cells are then sent to a bioreactor and expanded or cultured. In addition, T cells can be altered to have unique proteins called receptors. The receptors on the T cells are designed to recognize and target unwanted cells in the body, such as cancer cells. The modified T cells are cultured in a bioreactor to achieve a specific cell density and then delivered into the patient's body to fight cancer or other diseases. T cell therapy can also be referred to as adoptive T cell therapy or T cell transfer therapy, one example of which is called chimeric antigen receptor (CAR) T cell therapy. The use of T cells for adoptive T cell therapy or T cell transplantation therapy has recently seen a surge due to its great success in combating hematological disorders. In some embodiments, aspects of the invention can be used to monitor the health of T cells used in adoptive T cell therapy or T cell transplantation therapy. In some embodiments, aspects of the invention can be used to monitor T cell activation, T cell depletion, T cell metabolism, including starting materials and modified products.

[0087] NK cells are a type of cytotoxic lymphocyte that can seek out and destroy infected cells in the body. NK cells can display a very fast immune response. As a result, interest and popularity regarding the use of NK cells in anti-cancer therapy has increased significantly. However, there are only a limited number of NK cells in mammalian blood, and it is necessary to grow NK cells to a relatively high cell density in a bioreactor.

[0088] Culturing cells, such as T cells, NK cells, or other mammalian cells, typically requires a somewhat complicated process from inoculation to use in a patient. The disclosed assemblies and processes can be used to monitor cell metabolism at any point in the culture process to ensure that the cells are healthy and / or have a desired metabolic phenotype, and that the media in which the cells are growing contains optimized levels of nutrients. For example, the systems and processes can be used to make adjustments to ensure the metabolic fitness of the cells as they are growing.

[0089] In addition to immune cells, the metabolism of cancer cells can be monitored to provide understanding of which nutrients fuel cancer cells or how cancer cell metabolism can be targeted or regulated. For example, the assemblies and processes according to exemplary aspects of the present disclosure can reveal mechanisms or components that affect the metabolism of cancer cells to inhibit growth. The systems and processes of the present disclosure are also well suited for use in toxicology. For example, the processes and assemblies of the present disclosure can be used to detect mitochondrial disorders among potential treatments. For example, the risk of mitochondrial toxicity can be evaluated with high specificity and sensitivity. In this manner, the mechanism of action of some mitochondrial poisons can be identified.

[0090] The systems and processes according to exemplary aspects of the present disclosure can also be used to aid in the treatment of obesity, diabetes, and metabolic disorders by aiding in the discovery of relevant therapies. For example, the processes and systems can be used to measure the functional effects of genetic alterations on metabolic pathway components. Nutrients used in healthy and diseased cell models can be investigated. Additionally, fatty acid oxidation and glycolysis can be evaluated in different cell types, including stem cells.

[0091] Nevertheless, aspects of the present disclosure may be further understood by discussion of the following figures.

[0092] 1A-1C, a microtiter plate assembly 100 according to the present disclosure is shown. That is, the assembly 100 includes a magnetic plate adapter 101, a base 102, a lid 110, and a force distribution plate 103. As shown in FIGS. 1A-1C, the magnetic plate adapter 101 has a high degree of flatness in the x or horizontal direction, and the magnets 105 are mounted in an orthogonal direction (e.g., in the y direction, or extending vertically from the magnetic plate adapter 101). By orienting the magnets 105 in this manner, they can be positioned within the apron 107 of the base 102 such that the z-direction length l1 (as shown more clearly in FIG. 1B) or the longitudinal or x-direction length l2 at the base of the magnetic plate adapter 101 does not extend beyond the respective lengths of the base 102, yet still be able to interact with the force distribution plate 103 to provide forces as described above.

[0093] Additionally, as shown in Figure 1A, the magnetic plate adapter 101 and the force distribution plate 103 each include a number of apertures 109. Although Figure 1A shows the apertures 109 having different sizes and arrangements, it should be understood that in an aspect, the apertures 109 may be the same or different.

[0094] 1B shows the assembly of FIG. 1A in an assembled orientation, i.e., the magnetic plate adapter 101 is placed on a surface, and the base 102 is placed on the magnetic plate adapter 101 such that the magnets 105 are located under the apron 107. The lid 110 is then placed on the base, and the force distribution plate 103 is placed (aligned) on the lid 110 so that a material or magnet (not shown) magnetically contacts the magnets 105 of the magnetic plate adapter 101 to provide a strong connection in accordance with the present disclosure. As shown in FIG. 1B, the magnetic plate adapter 101 and the force distribution plate 103 have a length and width that is generally equal to or less than the base 102, and therefore no additional length or width is added to the assembly beyond the standard size of the base 102 and the lid 110.

[0095] In addition, FIG. 1C shows a cross section taken along line C-C' of FIG. 1B. As shown in FIG. 1C, the magnets 105 fit securely within the apron 107 so that they magnetically interact with the force distribution plate 103 to provide the necessary downward force as discussed above. Furthermore, as shown in FIG. 1C, the protrusions 112 have a very consistent height gap 154 ​​(the distance between the most distal point of the distal end 116 and the bottom surface (well bottom) 126 of the well). For example, as discussed above, by selecting one or more vertically magnetized magnets 105 with a force according to the ranges described above, the force distribution plate 103 is held in place by a downward force sufficient to reduce bowing and maintain the height gap described above. That is, even with the base and lid positioned between them as shown, this force can be transferred through the magnets 105 and the appropriate force can be maintained throughout the test as described above.

[0096] 2A and 2B, one embodiment of a base and lid for use in an assembly according to the present disclosure will be discussed in more detail. Specifically, as shown in FIG. 2A, a microtiter assembly 100 is shown having a base 102 having a plurality of wells 104, each having at least one sidewall 106 (more clearly shown in FIG. 2B) that defines a cavity 108. Additionally, FIG. 2A shows a lid 110 having a plurality of protrusions 112 that extend longitudinally from a proximal end 114 to a distal end 116 at substantially right angles from the lid 110. As shown more clearly in FIG. 2B, each protrusion 112 has a radial notch 122 that extends from the proximal end 114 to the distal end 116 of each protrusion 112. Additionally, each protrusion 112 has a beveled distal tip 124. In particular, as discussed above, the angled distal tip 124 is angled relative to the plane of the well bottom 126 and / or lid 110 according to the ranges discussed above.

[0097] As shown in FIG. 2A, a first row 118 of wells 102 have respective cavities 108 at least partially occupied by corresponding protrusions 112. While FIG. 2A shows an assembly with only a single row 118 of protrusions 112 attached to the lid 110, it should be understood from the above that the lid 110 can extend throughout the entire assembly and can include a number of protrusions 112 corresponding to the number of wells 104 in the respective base 102. Further, in one embodiment, each lid 110 can include one or more strips including protrusions 112, and each strip can be attached at one or more ends to a frame (shown more clearly in FIG. 4D below). For example, with respect to FIG. 2A, eleven additional strips having corresponding protrusions 112 can be attached to the frame such that each well 104 is occupied by a corresponding protrusion 112 to form a full 96-well microtiter plate 100.

[0098] 2B, the lid 110 is shown separate from the assembly 100 with projections 112 extending from a proximal end 114 to a distal end 116, each projection 112 having a radial notch 122 extending in at least one sidewall 128 of each projection. As shown in FIGS. 2A and 2B, the projections 112 are connected to supports 130. Additionally, while only a single row is shown in FIG. 2B, it should be understood that the supports 130 can be used to connect multiple lids 110 to a frame (not shown in FIGS. 2A and 2B). However, it should also be understood that in one aspect, the lid can be unitary and include multiple projections corresponding to respective bases.

[0099] Similarly, Figures 3A-3F show another embodiment similar to Figures 2A and 2B, where the lid 110 is in the form of a strip including eight protrusions 112. Figure 2A shows a bottom view of the lid 110. As shown in Figure 3A, the lid 100, in such an embodiment, can have a width w of about 12 mm or less, such as about 11 mm or less, such as about 10 mm or less, such as about 9 mm or less, such as about 5 mm or more, such as about 6 mm or more, such as about 7 mm or more, such as about 8 mm or more, or any range or value therebetween. For example, in one embodiment, the lid 110 has a width w of about 5 mm to about 12 mm, such as about 6 mm to about 11 mm, such as about 7 mm to about 9 mm, or any range or value therebetween.

[0100] 3A, which more clearly shows the apex 151 of the radial notch, the lid 110 can have a length 1 of about 100 mm or less, e.g., about 95 mm or less, e.g., about 90 mm or less, e.g., about 85 mm or less, e.g., about 80 mm or less, or e.g., about 60 mm or more, e.g., about 65 mm or more, e.g., about 70 mm or more, e.g., about 75 mm or more, or any range or value therebetween. For example, in one embodiment, the lid 110 has a length of about 60 mm to about 100 mm, e.g., about 65 mm to about 90 mm, e.g., about 70 mm to about 85 mm, or any range or value therebetween.

[0101] Similarly, with reference to Figure 3B (taken along section BB of Figure 2A) and Figure 2C, in one embodiment, the projection 112 has a height h1 from the stop tab 150 to the distal end 116, and a diameter d1, and the lid 110 has a height h2 from the stop tab receiver (not shown / shown more clearly in Figure 4A) to the well bottom surface 126, and a diameter d2. Using these dimensions, the volume of each well 104 and the volume of the corresponding projection 112 can be determined to determine the fluid volume displaced by the lid 110. Additionally, as shown in Figures 3B and 3C, the heights h1 and / or h2 can be measured from the centerline 111 of each projection.

[0102] For example, in one embodiment, h1 is about 15 mm or less, such as about 14 mm or less, such as about 13 mm or less, such as about 12 mm or less, such as about 11.5 mm or less, such as about 7 mm or more, such as about 8 mm or more, such as about 9 mm or more, such as about 10 mm or more, such as about 11 mm or more, or any range or value therebetween. For example, in one embodiment, the lid 110 has a height h1 of about 7 mm to about 15 mm, such as about 9 mm to about 13 mm, such as about 10 mm to about 12 mm, or any range or volume therebetween.

[0103] Additionally, h2 can be about 17.5 mm or less, such as about 16.5 mm or less, such as about 15.5 mm or less, such as about 14.5 mm or less, such as about 13.5 mm or less, such as about 13 mm or less, such as about 8.5 mm or more, such as about 9.5 mm or more, such as about 10 mm or more, such as about 10.5 mm or more, such as about 11.5 mm or more, such as about 12 mm or more, or any range or value therebetween. For example, in one embodiment, the lid 110 has a height h2 of about 8.5 mm to about 17.5 mm, such as about 10 mm to about 16.5 mm, such as about 11.5 mm to about 14.5 mm, or any range or volume therebetween. Furthermore, it should be understood that the ranges for h1 and / or h2 described above can be measured from the centerline 111 or the most distal point of the distal tip 127, as shown in FIG. 3B (distal-most point 127) and FIG. 2C (centerline 111).

[0104] Further, in one embodiment, d1 can be about 34.5 mm or less, for example, about 29 mm or less, for example, about 24 mm or less, for example, about 19 mm or less, for example, about 14 mm or less, for example, about 9 mm or less, for example, about 6.5 mm or less, for example, about 5 mm or less, for example, about 4.9 mm or less, for example, about 4.8 mm or less, for example, about 4.5 mm or less, for example, about 4 mm or less, for example, about 3.5 mm or less, for example, about 3 mm or less, for example, about 2.5 mm or less, for example, about 1.5 mm or less, for example, about 1 mm or more, for example, about 2 mm or more, for example, about 3 mm or more, for example, about 4 mm or more, for example, about 4.5 mm or more, for example, about 5 mm or more, or any range or value therebetween. For example, in one embodiment, the inner diameter d1 can be about 1 mm to about 34.5 mm, for example, about 2 mm to about 19 mm, for example, about 3 mm to about 9 mm, or any value or range therebetween.

[0105] Similarly, in one embodiment, d2 can be about 35.5 mm or less, e.g., about 30 mm or less, e.g., about 25 mm or less, e.g., about 20 mm or less, e.g., about 15 mm or less, e.g., about 10 mm or less, e.g., about 7.5 mm or less, e.g., about 5 mm or less, e.g., about 5 mm or more, e.g., about 5.5 mm or more, e.g., about 6 mm or more, e.g., about 6.3 mm or more, e.g., about 6.4 mm or more, e.g., about 6.45 mm or more. In one embodiment, the outer diameter d2 can be about 5 mm to about 35.5 mm, e.g., about 5.5 mm to about 25 mm, e.g., about 6 mm to about 20 mm, or any range or value therebetween. Furthermore, in one embodiment in which the protrusion 112 is tapered, it should be understood that the ranges for d1 and / or d2 described above can be measured at the widest or smallest diameter of d1 and / or d2, respectively.

[0106] 3B, in one embodiment, the protrusions 112 can be tapered towards the distal end 116 to provide further reduction in spillover without compromising the average wall clearance and to provide reduced evaporation. Thus, in one embodiment, one or more of the protrusions 112 have a centerline 111 that is generally perpendicular to the lid 110, and each protrusion has a taper 113 with an angle relative to the centerline 111 of about 2° or less, such as about 1.75° or less, such as about 1.5° or less, such as about 1.25° or less, such as about 1° or less, such as about 0.75° or less, or about 0.25° or more, such as about 0.4° or more, such as about 0.65° or more, or any range or value therebetween. For example, in one embodiment, one or more (or each) protrusion has a taper of from about 0.25° to about 2°, such as from about 0.35° to about 1.75°, such as from about 0.55° to about 1.5°, or any range or value therebetween.

[0107] 3D and 3E, where FIG. 3D is a top view of FIG. 3A and FIG. 3E is a cross-sectional view taken along line DD. An opening 115 is shown adjacent each radial notch 122. Nevertheless, as shown most clearly in FIG. 3E, the angled distal tip 124 is at an angle relative to the plane 119 of the lid 100, which may be generally planar as shown. Additionally, while the plane 119 of the lid 110 and the well bottom 126 were referred to above as parallel planes as shown in FIG. 3E, it should be understood that in one aspect, the well bottom 126 is angled at the same or a similar angle relative to the angled distal tip 124. As such, the well bottom 126 has a consistent thickness (not shown) throughout its cross-section. However, as described above, in one aspect, the well bottom surface 126 can lie in a plane that is generally parallel to the lid plane 119, such that the well bottom surface 126 has a thickness that increases toward the distal-most point of the sloped distal tip 124.

[0108] Finally, FIG. 3F illustrates a perspective view of the lid 110 of FIGS. 3A-3F, as shown, in an embodiment having an opening 115, which can be adjacent to a radial notch 122.

[0109] Further, as shown in FIG. 4A, in one embodiment, the lid 110 can be unitary and can have multiple projections 112 that are equal to the corresponding base 102 (not shown). Each projection 112 has a radial notch 122 extending in at least one sidewall 128 of each projection 112. Additionally, the lid 110 includes a frame 132 having a planar portion 134 and a lip 136. The lip 136 can include one or more beveled corners 138 or can have one or more square corners 140. In one embodiment, as shown in FIGS. 4A and 4B, two of these corners can be beveled to provide a reversible locking function to the lid 110 by providing a tighter fit and seal to maintain the lid 110 on the base (not shown) and further aid evaporation.

[0110] Additionally, the lip 136 can also have a length L3 that maintains SBS compatibility but extends further along the base 102 than the previous lid. For example, the length L3 can be about 10 mm or more, such as about 10.25 mm or more, such as about 10.5 mm or more, such as about 10.75 mm or more, such as about 11 mm or more, such as about 11.25 mm or more, such as about 11.5 mm or more, up to about 12 mm or less than about 12 mm, such as about 11.75 mm or less, such as about 11.7 mm or less, such as about 10 mm to about 12 mm, or about 10.25 mm to about 11.75 mm, such as about 10.5 mm to about 11.7 mm, or any range or value therebetween. That is, the present disclosure has found that a lip 136 having a length L3 within the ranges discussed above can further assist in reducing evaporation, which becomes more important when using reduced sample sizes.

[0111] 4B further illustrates the base 102 cooperating with the lid 110 of FIG. 4A. As shown, the base 102 includes a support 142 having a skirt 141, which includes a plurality of chambers or wells 104. In the embodiment shown, the base 102 also includes a beveled corner 138 that releasably attaches to the beveled corner 138 of the lid of FIG. 4A. As discussed above, the lid 110 of FIG. 4A can cooperate with each well 104 of FIG. 4B to occupy a portion of the cavity 108 formed by the well sidewalls 108. Although not shown, it should be understood that when a sample is present, the lid 110 can displace some or all of the gas located between the lid 108 and the sample (not shown), and in one embodiment, can evacuate all of the headspace of gas contained in each well 104, such that each projection 112 contacts the sample (not shown) contained in the respective well 104.

[0112] Additionally, FIG. 4C illustrates an assembly 100 according to the present disclosure in which the lid 110 (discussed above and shown more clearly in FIG. 4A ) is positioned onto the base 102 (shown more clearly in FIG. 4B above) such that each protrusion 112 is reversibly associated with a respective well 104 (shown more clearly in FIG. 4B ).

[0113] 4D shows another embodiment of an assembly 100 according to the present disclosure, in which the lid 110 is shown in two parts, a frame 152 and a cover 153. That is, as shown, an array of strips 156 including protrusions 112 are releasably attached to the frame 152. As such, the frame 152 can be formatted to include an appropriate number of strips 156 for the number of wells 104 included in each base 102. Furthermore, in such an embodiment, the cover 153 and / or the frame 152 can be formed from any of the substrate materials discussed above, or alternatively, the frame 152 can be formed from a rigid material such as a metal, which in one embodiment can be aluminum. Nevertheless, in one such embodiment, the cover 153 can be clear or matte.

[0114] 5A and 5B, a microtiter plate assembly 200 is shown having a lid 210 having a planar portion 234 and a lip 236. As discussed above, in one aspect, the frame (132 above) can be integral with the lid 210. Additionally, the lid 210 has a number of protrusions 212 that extend from a proximal end 214 adjacent the cover plate 210 to a distal end 216 having a beveled distal tip 224. As shown in FIG. 5A, the protrusions 212 can be generally hollow and can have an exterior sidewall 246 that contacts the sample and an interior sidewall 248 located within each protrusion. However, it should be understood that even if the protrusions are hollow, the radial notches 222 do not penetrate through the exterior sidewall 246 into the interior sidewall 248. In one embodiment, for example, protrusion 212 is hollow when formed by injection molding, although it should be understood that any method known in the art, such as cutting, may be used.

[0115] In addition, FIG. 5A more clearly illustrates the stop tabs 250. As discussed above, the stop tabs 250 can be used to provide very precise height and wall clearances in conjunction with the radial notches 222 and angled distal tips 224, as discussed in more detail in connection with FIG. 5B. That is, the stop tabs 250 contact a portion of the base 202 to position the projections 212 at a specific height to maintain the required height and wall clearances (as shown more clearly in FIG. 5B). In one aspect, the stop tabs 250 can contact a continuous portion of the base 202, or alternatively, can contact the stop tab receivers 258. Nevertheless, as shown, the microtiter plate assembly 200 also includes a plurality of wells 244 configured to reversibly releasably receive the projections 212.

[0116] 5A, the projections 212 have a height h1 from the stop tabs 250 to the distal end 216, and a diameter d1, and the lid 210 has a height h2 from the height of the stop tabs 250 to the well bottom 226, and a diameter d2. Using these dimensions, the volume of each well 204 and the volume of the corresponding projections 212 can be determined to determine the fluid volume displaced by the lid 210.

[0117] Figure 5B includes a close-up view of the distal end 216 of the projection 212 of Figure 5A. That is, as shown in Figure 5B, the distal end 216 of the projection 212 has a distal tip 224 that is angled relative to the well bottom 226 (e.g., the underside of the interior of the well cavity). Additionally, Figure 5B more clearly shows the sample 252, height gap 254, wall gap 256, radial notch 222, apex 251, and radial notch gap 253, as discussed above. That is, as shown, a small but precise height gap 254 (e.g., the distance between the distal-most portion of the projection 212 / the portion of the projection 212 closest to the well bottom 226) is close to the well bottom 226, allowing for sensitive measurements without contacting the well bottom 226, which could inhibit cell growth and seeding. Additionally, as shown, the wall gap 256 is made very small to allow the projection 212 to be removed from the well 244 while still directing any air bubbles into the radial notch 222 .

[0118] 5B further illustrates that the radial notch 222 is located adjacent to the highest portion of the angled distal tip 224 (e.g., the portion of the distal end 216 having the shortest distance from the proximal end 224, or the portion of the angled distal tip 224 furthest from the well bottom 226). In this way, the angle of the angled distal tip 224 can guide the air bubbles into the radial notch 222 so that the air bubbles can be efficiently expelled from the sample 252. As shown in FIG. 4B, the sample 252 has a height h that is slightly higher than the height of the angled portion of the distal tip 224 plus the height gap 254. In this way, the sample 252 can enter the radial notch 222, thus preventing overflow, but completely eliminating the head space.

[0119] 5C illustrates an embodiment of the present disclosure in which protrusion 212 includes two radial notches 222 and two angled surfaces 225 on angled distal tip 224 having a distal-most end adjacent centerline C and a highest end adjacent each notch 222. Additionally, as shown, protrusion 212 includes spot surface 260 that is generally perpendicular to well bottom surface 226.

[0120] Meanwhile, Figures 9A-9C show additional embodiments of lids and force distribution plates for use in assemblies according to the present disclosure. That is, as shown in Figure 9A, a microtiter plate assembly 100 is shown having a base 102 having a plurality of wells 104. Additionally, Figure 9A shows a lid 110 having a plurality of protrusions (not shown) extending longitudinally from the lid 110, as described above with respect to Figure 1A. The lid 110 is shown in top view in Figure 9B. In particular, the lid 110 can include a plurality of spacers 160 disposed on an outer surface 167 thereof. The spacers 160 can be located at a periphery 166 of the lid 110 and / or at a center 168 of the lid 110, or at any suitable location. The inventors have found that the use of such spacers 160 can prevent warping of the lid 110 by increasing the downward pressure exerted on the base 102 through the lid 110, providing more accurate test results, achieving sufficient sample volume consistency across the base 102, and resulting in a more consistent rate of oxygen depletion across the base 102.

[0121] 9B, multiple spacers 160 can be present in a first outer region 161 of the lid 110, multiple spacers 160 can be present in a second outer region 162 of the lid 110, and a single spacer 160 can be located in a central region 163 of the lid 110. However, it should be understood that additional spacers 160 can be located in any of the regions 160, 161, or 162 to achieve reduced warpage of the lid 110. Furthermore, the spacers 160 can be injection molded during formation of the lid 110 and can be formed from the same material as the remainder of the lid 110.

[0122] It should further be appreciated that the spacers 160 can have varying thicknesses in the y-direction to provide uniformity and reduced warping of the lid 110. However, in other embodiments, the spacers 160 can each have the same thickness. For example, in some embodiments, the spacers 160 can each have a thickness of about 100 micrometers to about 500 micrometers, such as about 125 micrometers to about 475 micrometers, such as about 150 micrometers to about 450 micrometers.

[0123] In one particular embodiment, the thickness of the spacer 160 in the central region 163 can be greater than the thickness of the spacer 160 in the first outer region 161 and the second outer region 162 to provide increased downward pressure at the center of the lid 110 where greater warpage may occur. In such an embodiment, the spacer 160 in the central region 163 can have a thickness of about 175 micrometers to about 500 micrometers, such as about 200 micrometers to about 475 micrometers, such as about 225 micrometers to about 450 micrometers, and the spacer 160 in the first outer region 161 and the second outer region 162 can have a thickness of about 100 micrometers to about 250 micrometers, such as about 125 micrometers to about 225 micrometers, such as about 150 micrometers to about 200 micrometers.

[0124] As shown in FIG. 10, a comparison of the coefficient of variation of oxygen depletion measurements in wells of the lid 110 of the microtiter plate assembly 100 of FIGS. 9A and 9B utilizing multiple spacers 160 having various thicknesses compared to a lid 110 without spacers shows that the coefficient of variation (% CV) is reduced when multiple spacers 160 are utilized compared to when no spacers are utilized.

[0125] 9C, in addition to or instead of the lid 110, the force distribution plate 103 may also include a number of spacers 170 disposed on an inner surface 177 of the force distribution plate 103. The spacers 170 may be located at the periphery 176 of the force distribution plate 103 and / or at the center 178 of the plate 103, or at any suitable location. The inventors have found that the use of such spacers 170 can prevent warping of the lid 110 and provide more accurate test results by increasing the downward pressure exerted on the base 102 through the plate 103. As shown in FIG. 9C, a number of spacers 170 may be present in a first outer region 171 of the plate 103, a number of spacers 170 may be present in a second outer region 172 of the plate 103, and a single spacer 170 may be located in a central region 173 of the plate 103. However, it should be understood that additional spacers 170 could be located in either areas 170, 171, or 172 to achieve reduced warping of the lid 110 through the plate 103, achieve sufficient sample volume consistency across the base 102, and result in a more consistent rate of oxygen depletion across the base 102.

[0126] It should further be appreciated that the spacers 170 may have varying thicknesses in the y-direction to provide uniformity and reduced warping of the lid 110 through the plate 103. However, in other embodiments, the spacers 170 may each have the same thickness. For example, in some embodiments, the spacers 170 may each have a thickness of about 100 micrometers to about 500 micrometers, such as about 125 micrometers to about 475 micrometers, such as about 150 micrometers to about 450 micrometers. In one particular embodiment, the thickness of the spacers 170 in the center region 173 may be greater than the thickness of the spacers 170 in the first outer region 161 and the second outer region 172 to provide increased downward pressure at the center of the lid 110 where greater warping may occur. In such an embodiment, the spacer 160 in the central region can have a thickness of about 175 micrometers to about 500 micrometers, such as about 200 micrometers to about 475 micrometers, such as about 225 micrometers to about 450 micrometers, and the spacer 170 in the first outer region 171 and the second outer region 172 can have a thickness of about 100 micrometers to about 250 micrometers, such as about 125 micrometers to about 225 micrometers, such as about 150 micrometers to about 200 micrometers.

[0127] Aspects of the present disclosure will be further understood from the following non-limiting examples.

[0128] [Example 1] A microtiter plate assembly according to Figures 1A-1C was formed with a magnetic plate adapter having 24 apertures and two magnets, the two magnets being oriented perpendicular to the magnetic plate adapter and magnetized horizontally. The magnets were positioned near the center points of both ends of the magnetic plate adapter spaced apart longitudinally as shown in Figure 1A. Each magnet had a magnetic force of about 4 kgf for a total magnetic force of about 8.75 kgf. A base and lid as shown in Figure 1A was provided on top of the magnetic plate adapter, and a force distribution plate formed of coated steel with black zinc oxide having 96 circular apertures was placed on top of the lid.

[0129] The stability of the lid, microplate, and adapter assembly was evaluated by comparing the average MitoXpress Xtra signal from the outer to inner rows of wells (e.g., the outer rows are the rows on the outer edge of the assembly, such as the first and last rows, and the inner rows are included between the outer rows on either side). MitoXpress Xtra reagent is dissolved within the sample volume, so an increased signal should indicate an increased sample volume. The acquired data was evaluated by calculating the mean fluorescent signal and standard deviation from each row of wells (e.g., A1-A12, B2-B12, etc.) on the 96-well plate. The mean signal is then expressed as a % of the mean signal of the center rows (C, D, E, and F). This allowed for a comparison of the differences between the outer edge and inner / center wells / samples of the assembly.

[0130] Signals were measured at 0, 30, 60, 90, and 120 minutes to assess changes in stability (changes in well volume related to post / lid movement) over time.

[0131] A signal variation of less than ±10% indicates a very stable signal / assembly.

[0132] As shown in the examples below, in the absence of a downward pressure device, signal changes in the outer rows can exceed 25-30% due to the corners of the lid moving or lifting during the assay. When the lid is assembled as one unit, this also affects adjacent wells, resulting in consistent measurements from only a few wells in the center of the plate.

[0133] Throughout this example, temperature was maintained at 37° C. and fluorescence was determined using a BMG Clariostar and standard MitoXpress Xtra assay protocol.

[0134] After the assemblies were assembled, they were subjected to warpage testing as defined above and the RFU was measured over time, as shown in Figure 1B. As shown in Figure 6, the assemblies of the present disclosure exhibited excellent warpage even at 120 minutes.

[0135] [Example 2] Example 2 was performed in a similar manner to Example 1. In addition, an oil-in-headspace sample and a sample with seven permanently attached screws holding the assembly together were tested using the warpage test defined above along with the two samples for a 90 minute runtime. That is, as discussed above, an increased signal over time indicates upward movement of the lid and therefore an increased sample volume, which is caused by warping of the lid. As shown in FIG. 7, the assembly according to the present disclosure exhibited less than 10% warpage.

[0136] [Example 3] The assembly of Example 1 was tested for sensitivity to a standard oil-in-headspace assay. For both the sample assay assembly and the oil-in-headspace assay, seeding densities of 10,000, 35,000, and 70,000 were tested using the MitoXpress Xtra and CLARIOstar plate readers described above to determine sensitivity to the oxygen consumption rate of C2C12 cells. This testing yielded the following results:

[0137] [Table 1]

[0138] As shown in Table 1, the assembly according to the present disclosure was capable of detecting oxygen consumption rates below confluence in 30-45 minutes.

[0139] [Example 4] An assembly according to Example 1 was formed, with the plate adapter and force distribution plate formed from steel. A Starrett Granite Surface Plate (2720-0M) with electronic digital readout was used to measure the deflection point of the outer row under stress testing according to the following:

[0140] As discussed above, the microtiter plate assembly was assembled by placing the well / microplate assembly onto a plate adapter containing one or more magnets and placing a steel force distribution plate onto the lid of the well / microplate assembly. After assembly at room temperature, the microtiter plate assembly was placed into a 37° C. heating environment and left for 2 hours. After the 2 hours of heating, the assembly was removed from the heat, disassembled, and allowed to cool to room temperature.

[0141] After cooling, the plate adapter and force distribution plate were measured to determine flatness at nine points shown in Figures 8A and 8B by measuring the distance between the bottom surface of each plate and the measurement surface at each of the locations. Flatness measurements were also recorded under deflection, where the deflection occurred at a point opposite the measurement location (e.g., for measurements at LT, the deflection was RB). The heating, cooling, and measurement were then repeated nine more times for a total of 10 cycles. The average deflection measurements are shown in Figures 8D and 8D. As shown, even utilizing the extremely sensitive Starrett system, the plate adapter and force distribution plate exhibited excellent flatness over the stress test cycles.

[0142] These and other modifications and variations to the present disclosure may be implemented by those skilled in the art without departing from the spirit and scope of the present invention as more particularly set forth in the appended claims. In addition, it should 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 by way of example only and is not intended to limit the invention as further set forth in such appended claims.

Claims

1. 1. A microtiter plate assembly comprising: a base including a plurality of wells in at least one outer row and at least one inner row; a lid including a plurality of protrusions corresponding to the plurality of wells; A magnetic plate adapter; A force distribution plate; Equipped with At least a portion of the force distribution plate, the magnetic plate adapter, or both the magnetic plate adapter and the force distribution plate, formed from a magnetic or paramagnetic material; having a magnetic or paramagnetic material disposed on at least a portion of the a magnetic material attached to said at least a portion; the at least one magnet provides sufficient force to reduce warping of the lid; the microtiter plate assembly exhibits a warp rate of about 30% or less, the warp rate being defined as a signal change rate of an average fluorescent signal of the at least one outer row relative to an average fluorescent signal of the at least one inner row; Microtiter plate assembly.

2. 1. A microtiter plate assembly comprising: a base including a plurality of wells spaced apart such that each well is adjacent to a second well; a lid including a plurality of spaced apart projections corresponding to the array of wells such that each projection is adjacent to a second projection; a magnetic plate adapter having at least one magnet; A force distribution plate; Equipped with At least a portion of the force distribution plate, the magnetic plate adapter, or both the magnetic plate adapter and the force distribution plate, formed from a magnetic or paramagnetic material; having a magnetic or paramagnetic material disposed on at least a portion of the a magnetic material attached to said at least a portion; the plurality of projections extend from a proximal end adjacent the lid to a distal end; a height gap is defined between a distal end of each projection and a bottom well surface of each well; The variation rate of the height gap between the protrusion and the well and the adjacent second protrusion and the second well is about 10% or less. Microtiter plate assembly.

3. 3. The microtiter plate assembly of claim 1 or claim 2, wherein the force distribution plate, the magnetic plate adapter, or both the magnetic plate adapter and the force distribution plate provide at least about 4 kilogram force (kgf) to the lid.

4. The microtiter plate assembly of any one of claims 1 to 3, wherein the microtiter plate assembly exhibits a warpage rate of about 15% or less.

5. A microtiter plate assembly as described in any one of claims 1 to 4, wherein the longitudinal length of at least one of the magnetic plate adapter and the force distribution plate is equal to or shorter than the longitudinal length of the base.

6. The microtiter plate assembly of any one of claims 1 to 5, wherein the base includes an apron, the apron configured to releasably house the at least one magnet.

7. A microtiter plate assembly according to any one of claims 1 to 6, wherein the microtiter plate assembly is adapted to be compatible with a plate reader and / or is Society for Biomolecular Screening (SBS) compliant.

8. The microtiter plate assembly of any one of claims 1 to 7, wherein said at least one magnet is magnetized vertically.

9. The microtiter plate assembly of any one of claims 1 to 8, wherein at least one of the magnetic plate adapter and the force distribution plate has a thickness of about 12 mm or less.

10. The microtiter plate assembly of any one of claims 1 to 9, wherein at least one of the magnetic plate adapter and the force distribution plate has a weight of about 600 g or less.

11. The microtiter plate assembly of any one of claims 1 to 10, wherein at least one of the magnetic plate adapter and the force distribution plate includes one or more apertures.

12. The microtiter plate assembly of any one of claims 1 to 11, wherein the microtiter plate assembly comprises 96 wells and 96 corresponding protrusions.

13. The microtiter plate assembly of any one of claims 1 to 12, wherein at least one of the magnetic plate adapter and the force distribution plate is formed from aluminum, steel, a combination thereof, or an alloy or derivative thereof.

14. Each protrusion is approximately 0.8 mm ２ A microtiter plate assembly according to any one of claims 1 to 13, comprising a radial notching having a radial cross-sectional area of ​​at least 1 mm.

15. A microtiter plate assembly according to any preceding claim, wherein a distal end of each projection is inclined at an angle of about 5° or greater relative to the plane of the lid.

16. At least a portion of the microtiter plate assembly is formed from an acrylate polymer, or at least a portion of the microtiter plate assembly is resistant to about 15 cm ３ / m ２ A microtiter plate assembly according to any one of claims 1 to 15, which is coated with a polymer having an oxygen permeability of 24 hours.

17. The microtiter plate assembly of any one of claims 1 to 16, wherein at least one of the lid and the force distribution plate includes a plurality of spacers.

18. A method for measuring an oxygen consumption rate using the microtiter plate assembly according to any one of claims 1 to 17, comprising the steps of: placing a sample and an oxygen-sensitive phosphorescent probe into one of said plurality of wells; contacting the sample with a corresponding one of the protrusions; measuring said oxygen consumption rate by a fluorescent plate reader; A method comprising:

19. 20. The method of claim 18, wherein the sample has a seeding density of less than 70,000 cells and the measurement is performed in 45 minutes or less.

20. 20. The method of claim 18 or claim 19, wherein the seeding density is controlled at sub-confluence of the selected cell type.

21. The method according to any one of claims 18 to 20, wherein the sample volume is equal to or less than 100 μL, preferably the sample volume is equal to or less than 70 μL.

Images