Headspace Eliminating Microtiter Plate Lid

Abstract

A microtiter plate assembly is disclosed that includes a base having a plurality of wells and a lid having a plurality of protrusions corresponding to the plurality of wells, each protrusion including a radial notch and a beveled distal tip, providing a bubble-free seal, reduced oxygen back diffusion, and increased sensitivity even with small sample volumes.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 328,894, filed April 8, 2022, which is incorporated by reference in its entirety. [Background technology]

[0002] Test tubes and microtiter plates are commonly used with oxygen-sensitive photoluminescent probes to measure and monitor the aerobic activity of a sample by measuring and monitoring the oxygen concentration within the tube or well. This requires sealing the sample from fluid communication with the surrounding environment, which is often accomplished by providing an oil layer above the sample and interrogating the oxygen-sensitive photoluminescent probe within the sample through the oil layer. The use of an oil layer to seal the sample provides the added benefit of limiting the presence of a gaseous headspace between the sample and the oil layer. A gaseous headspace trapped below an oxygen barrier layer is known to slow down the detection of changes in oxygen concentration due to the relatively large supply of oxygen available within such a gaseous headspace.

[0003] While generally effective in sealing the sample from direct fluid communication with the surrounding environment and limiting the presence of a gaseous headspace below the oil layer, the oil layer is not a particularly effective oxygen barrier, is difficult to deploy properly and consistently, and is labor intensive. However, utilizing oil as a headspace elimination mechanism requires increased sample volume to avoid a reduction in optical path length (which may be caused, for example, by oil contacting the bottom of the cell culture due to meniscus formation), which reduces sensitivity below that required for many measurement devices. For example, such high sample volumes require correspondingly high seeding densities, making it difficult to study cells at subconfluent densities, which is further exacerbated when studying cells with altered metabolism at confluence. Furthermore, oil as a headspace elimination mechanism also requires a step of applying oil to each well, which not only creates the potential for contamination and error, but also precludes the possibility of performing post-assay testing.

[0004] Other devices, such as microtiter lids, have been proposed to allow increased visibility and control sample spillover, however, the proposed devices are not designed for metabolic interrogation purposes in that they do not limit oxygen ingress into the sample, nor do they adequately dissipate bubbles from the enclosed sample.

[0005] Thus, although provisions have been proposed to eliminate the gaseous headspace in test tubes and microtiter plates, there remains a need for improved systems and methods suitable for measuring and monitoring oxygen consumption in which both headspace and bubble formation are reduced. Summary of the Invention

[0006] The present disclosure is generally directed to a microtiter plate assembly including a base and a lid. The base includes at least one well, and the lid includes at least one translucent protrusion corresponding to the at least one well. The at least one translucent protrusion extends from a proximal end adjacent the lid to a distal end having a radial notch and an oblique tip, the oblique tip having an angle of about 5° or more relative to the plane of the lid, and a wall clearance is defined between a sidewall of the at least one protrusion and a sidewall of the at least one corresponding well. Furthermore, a radial notch clearance is defined between an apex of the radial notch and a sidewall of the corresponding well, the wall clearance being 50% or less of the radial notch clearance.

[0007] The present disclosure is also generally directed to a microtiter plate assembly including a base and a lid. The base includes a plurality of wells spaced apart in an array, each well adjacent to a second well, and the lid includes a plurality of semi-transparent protrusions spaced apart in an array corresponding to the array of the plurality of wells, each protrusion adjacent to a second protrusion. In addition, the plurality of semi-transparent protrusions extend from a proximal end adjacent to the lid to a distal end having a radial notch and a beveled tip, the beveled tip having an angle of about 5° or more with respect to the plane of the lid. Furthermore, a height clearance is defined between the distal-most point of the distal end of each protrusion and a well base of the respective well, and the percent variation in height clearance between the protrusion and the well and between the adjacent second protrusion and the second well is about 10% or less.

[0008] In a further embodiment, at least a portion of the assembly is about 600 cm 3 / m 2 and / or at least a portion of the assembly is formed from a substrate material having an oxygen transmission rate of about 120 cm 3 / m 2 In one embodiment, at least a portion of the assembly is coated with a polymer or ceramic compound having an oxygen transmission rate of 1 / 24 hours or less. 2 O 3 (Aluminum oxide), SiO 2(silicon dioxide), silicon oxide, silicon nitride, silicon oxynitride, aluminum oxide, Al alternating with polyacrylates 2 O 3 , or a combination thereof.

[0009] In a still further aspect, the radial notch is about 0.7 mm 2 or more, and preferably the radial cutout is about 1.25 mm 2 In one embodiment, the radius of the radial notch has a length of about 5% to about 30% of the length of the radius of the cross section of the protrusion including the radial notch at the proximal end of the protrusion, the distal end of the protrusion, or at both the proximal and distal ends of the protrusion. In still a further embodiment, at least one protrusion has a proximal end radius and a distal end radius, the distal end radius being 2% to about 15% less than the proximal end radius.

[0010] Moreover, in one embodiment, the assembly is configured to measure oxygen consumption of a fluid sample having a volume of 100 μL or less. In still a further embodiment, the assembly is configured to measure oxygen depletion of a sample having a seeding density of 250,000 cells or less for a period of 45 minutes or less.

[0011] In one embodiment, the lid is in reversible association with the base. In another embodiment, the lid is milled, extruded, injection molded, or any combination thereof. Furthermore, in one embodiment, the assembly includes about 1 to about 384 wells and about 1 to about 384 corresponding projections. In another embodiment, the lid is a single piece. Additionally or alternatively, in one embodiment, the lid is formed from a frame and one or more rows including a plurality of translucent projections attached to the frame. In another embodiment, the lid includes eight rows, each row including twelve corresponding projections. Furthermore, in one embodiment, at least a portion of the assembly is formed from an acrylate polymer. Furthermore, in another embodiment, the lid can include a plurality of spacers.

[0012] The present disclosure is also generally directed to a method of measuring oxygen deficiency utilizing an assembly according to any one or more of the embodiments discussed above, the method including placing a sample and an oxygen-sensitive phosphorescent probe into at least one well, contacting the sample with the corresponding protrusion, and measuring oxygen deficiency using a fluorescent plate reader.

[0013] In one embodiment, the method comprises measuring extracellular acidification.Furthermore, in one embodiment, the oxygen-sensitive phosphorescent probe is a solid sensor coated on the distal end of the protrusion, or the oxygen-sensitive phosphorescent probe is a particle.In yet another embodiment, the sample has a volume of 100 μL or less, preferably the sample volume is 70 μL or less.

[0014] Other features and aspects of the disclosure are discussed in further detail below.

[0015] A full and enabling disclosure of the present disclosure is more particularly set forth in the remainder of the specification, including reference to the accompanying drawings. [Brief description of the drawings]

[0016] [Figure 1A] FIG. 1 illustrates one embodiment of a microtiter plate assembly according to the present disclosure. [Figure 1B] FIG. 1B illustrates an embodiment of a microtiter plate lid according to FIG. 1A. [Figure 2A] FIG. 2 is a top down view of another embodiment of a microtiter plate lid according to the present disclosure. [Figure 2B] FIG. 2B illustrates the microtiter plate lid of FIG. 2A along cross section BB. [Figure 2C] FIG. 2B is a side perspective view of the microtiter plate lid of FIG. 2A. [Figure 2D] FIG. 2B is a bottom-up view of the microtiter plate lid of FIG. 2A. [Figure 2E]FIG. 2C is a cross-sectional view of FIG. 2D along DD. [Figure 2F] FIG. 2B is a perspective view of the microtiter plate lid of FIG. 2A. [Figure 3A] FIG. 1 illustrates another embodiment of a microtiter plate lid according to the present disclosure. [Figure 3B] FIG. 3B illustrates a microtiter plate base that corresponds to the microtiter plate lid of FIG. 3A. [Figure 3C] FIG. 3C illustrates an embodiment of the microtiter plate lid of FIG. 3A in releasable association with the microtiter plate base of FIG. 3B. [Figure 3D] FIG. 1 illustrates an embodiment of a microtiter assembly according to the present disclosure. [Figure 4A] 1 illustrates a further embodiment of a microtiter plate assembly according to the present disclosure. [Figure 4B] FIG. 4B is an enlarged perspective view of the distal end of the protrusion of FIG. 4A. [Figure 4C] FIG. 13 is an enlarged perspective view of a distal end of a prong according to the present disclosure. [Figure 5A] 1 is a graph of reoxygenation over time according to Example 1. [Figure 5B] 1 is a graph of reoxygenation over time according to Example 1. [Figure 5C] 1 is a graph of reoxygenation over time according to Example 1. [Figure 6] 1 is a graph of oxygen consumption over time according to Example 2. [Figure 7] 1 is a graph of oxygen consumption as a function of cell seeding density according to Example 3. [Figure 8] 1 is a graph of oxygen depletion over time according to Example 4. [Figure 9] 1 is a graph of pH calibration according to Example 5. [Figure 10A] 1 is a graph showing the measured pH and oxygen depletion according to Example 5. [Figure 10B] 1 is a graph showing the measured pH and oxygen depletion according to Example 5. [Figure 11A]FIG. 1 illustrates another embodiment of a microtiter plate assembly according to the present disclosure. [Figure 11B] FIG. 1 illustrates another embodiment of a microtiter plate assembly according to the present disclosure. [Figure 12] 11C is a graph showing a comparison of the coefficient of variation of oxygen depletion measurements in wells of the lids of the microtiter plate assemblies of FIGS. 11A and 11B utilizing spacers having various thicknesses compared to lids without spacers. [Figure 13] FIG. 13 shows images to demonstrate cell survival of cells cultured in a plate assembly of the present disclosure of a control sample of culture medium and culture medium containing tamoxifen after 1 hour and 24 hours. [Figure 14] 1 is a graph illustrating oxygen consumption measured for cells grown in culture medium containing tamoxifen after 1 hour and 24 hours. [Figure 15] FIG. 13 shows images taken of cells cultured in a plate assembly of the present disclosure for cells grown in a control sample of culture medium and for cells grown in culture medium containing rotenone, antimycin, and oligomycin. [Figure 16] 1 is a graph illustrating oxygen consumption measured for cells grown in culture medium containing rotenone, antimycin, and oligomycin compared to control (untreated) medium samples. [Figure 17] FIG. 13 shows a comparison of images taken when a lid of the plate assembly of the present disclosure is utilized compared to when no lid is utilized (open) when measuring JC-1. [Figure 18] FIG. 1 shows a comparison of images taken before and after treatment with FCCP and oligomycin using a plate assembly of the present disclosure. [Figure 19] 1 is a graph illustrating oxygen consumption measured for cells grown in culture medium containing FCCP and oligomycin compared to cells grown in control (untreated) culture medium. [Figure 20]1 is a graph illustrating the dynamic JC-1 ratio measured for cells grown in culture medium containing FCCP and oligomycin compared to cells grown in control (untreated) culture medium. [Figure 21] 1 is a graph illustrating the ability to measure respiration of cells grown in suspension and treated with various compounds utilizing the plate assembly of the present disclosure. [Figure 22A] 13 is an image illustrating that suspension cells cultured in a plate assembly of the present disclosure can be spiked with JC-1 and imaged. [Figure 22B] 13 is an image illustrating that suspension cells cultured in a plate assembly of the present disclosure can be spiked with JC-1 and imaged. [Figure 22C] 13 is an image illustrating that suspension cells cultured in a plate assembly of the present disclosure can be spiked with JC-1 and imaged. [Figure 22D] 13 is an image illustrating that suspension cells cultured in a plate assembly of the present disclosure can be spiked with JC-1 and imaged. [Diagram 23] FIG. 25 shows JC-1 ratios calculated for the imaged cells in FIGS. 22A-22D. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Repeat use of reference characters in the present specification and drawings is intended to represent same or analogous features or elements of the invention.

[0018] Definitions and Test Methods The oxygen concentration can be determined or indirectly interrogated by placing an oxygen-sensitive photoluminescent probe and a fluid test sample in multiple wells in a microtiter plate, exposing the oxygen-sensitive photoluminescent probe in each well to excitation radiation that passes through the projections extending therein or through the well bottom to create excited oxygen-sensitive photoluminescent material, measuring the radiation emitted by the excited oxygen-sensitive photoluminescent material that passes through the projections and the well bottom, and converting the measured radiation to a target analyte concentration based on known conversion algorithms, or by measuring the probe / sensor signal in either intensity or lifetime mode to determine the oxygen concentration in each well of the coated microtiter plate. In one embodiment, a suitable photoluminescent probe for such measurements is MitoXpress® Xtra available from Agilent Technologies. Suitable instruments 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, Ortenberg, Germany, and the Synergy HTX from BioTek, an Agilent Technologies company. However, it should be understood that the photoluminescent material may also include an indicator dye incorporated within an oxygen-permeable polymer matrix, and electrochemical sensors may also be used to determine oxygen consumption. Oxygen consumption rate (OCR), which may also be referred to as oxygen depletion rate, as used herein, is the rate at which metabolic substances (O 2 ) are consumed from a medium sample. 2 ), which can then be reported in the form of a rate (change in analyte over time) or by measuring or indirectly assessing the analyte concentration at a preselected time point (end point). Conversely, oxygen production, which results in an increase in oxygen concentration, can also be determined by measuring the increase in oxygen over time.

[0019] The change in oxygen consumption can be determined in a closed or non-closed system. In one embodiment, the definition of OCR includes that the oxygen consumption is not determined in a closed system, e.g., the system allows oxygen back diffusion or substantial oxygen back diffusion to the sample, or that the oxygen consumption is the oxygen deficiency in the sample corrected for oxygen back diffusion to the sample, or that the oxygen consumption is the oxygen deficiency without being corrected for oxygen back diffusion to the sample, or that the oxygen consumption is determined in a closed system, e.g., a system that does not allow oxygen back diffusion or substantial oxygen back diffusion to the sample, or that the oxygen consumption is equal to or substantially equal to the oxygen deficiency in the sample. Furthermore, in one embodiment, the oxygen consumption is determined, e.g., inferred, directly or indirectly, e.g., from a measured oxygen gradient in the test well or by measuring oxygen at preselected time points.

[0020] Extracellular acidification rate (ECAR), as used herein, can be determined using a baseline or initial value of proton flux for a cell sample, for example, a value based on measuring proton flux for a cell sample before forming a reaction mixture, and determining the proton flux rate after forming a reaction mixture. For example, (ECAR), as used herein, can be calculated by sensing the metabolic substance (H+) that is either consumed or produced in a medium sample, which can 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 ECAR can be determined in a closed or non-closed system.

[0021] With regard to data processing, in one embodiment, signals such as the MitoXpress® signal can be output in relative fluorescence units (RFU) and processed to adjust for the nonlinear oxygen response of the MitoXpress® probe and any potential temperature equilibration within the assay. This is accomplished by taking the natural logarithm of the RFU signal and subtracting the natural logarithm of the signal control at each time point. The oxygen calibration of the MitoXpress® probe has an exponential fit, which makes it a simple way to linearize the signal. The level of temperature equilibration and drift in the probe signal is captured in the signal control and accounted for through subtraction. This ensures that a larger portion of the oxygen depletion trace is linear, resulting in a reduced coefficient of variation (%CV) for more robust data analysis and assays. This is especially important for higher rates in the RFU or lifetime scale, where several measurement points fall within the linear range of the curve. The signal can be further processed by normalizing to the first readout of the assay to better evaluate and compare compound responses.

[0022] As used herein, "semi-transparent" or "semi-transparent" refers to the light transmittance or optical density of a material that is in the range between transparent and opaque. Generally speaking, a translucent material allows light to pass diffusely. A material that is translucent allows greater levels of electromagnetic radiation in the visible spectrum to pass through it than an opaque or substantially opaque material, but allows lower levels of electromagnetic radiation in the visible spectrum to pass through it than a transparent or substantially transparent material. A translucent material may still provide protection from phototoxicity while still allowing detection of cells. As a result, cells cannot be observed from above the plate.

[0023] As used herein, the terms "about," "approximately," or "generally," when used to modify a value, indicate that the value may be increased or decreased, such as by 10%, such as by 7.5%, such as by 5%, such as by 4%, such as by 3%, such as by 2%, such as by 1%, and still remain within the disclosed aspects. Furthermore, the term "substantially free," when used to describe the amount of a substance in a material, is not limited to total or complete absence, but may correspond to the absence of any apparent or detectable amount of the recited 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 may 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.

[0024] Detailed Description It will be understood by those skilled in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broad aspects of the present disclosure.

[0025] Generally speaking, the present disclosure is directed to a microtiter assembly including a lid having multiple protrusions that eliminates the headspace present in existing microtiter assemblies, and a base having multiple reciprocating chambers (e.g., wells) corresponding to each of the protrusions. Thus, in such a manner, the headspace eliminating microtiter lid of the present disclosure may also improve oxygen back diffusion and ingress while allowing for a reduction in sample size and fill volume. The present disclosure has unexpectedly discovered that a microtiter plate assembly having multiple protrusions extending longitudinally from the lid, each protrusion having a combination of radial notches and height clearances within a particular range in combination with an angled distal end, allows ambient headspace oxygen to be reduced or eliminated, including completely eliminated, and limits oxygen back diffusion (oxygen ingress) while allowing for a reduction in sample size.

[0026] In particular, in one embodiment, the present disclosure has discovered that by utilizing specifically sized radial notches extending lengthwise from the distal end to the proximal end of each protruding portion, air bubbles present within the sample chamber (e.g., well) are allowed to escape while preventing sample overflow. In one embodiment, the radial notches, which will be discussed in more detail in connection with the figures below, are approximately 0.575 mm. 2 The above is an example of approximately 0.585 mm. 2 The above is an example of approximately 0.595 mm. 2 The above is an example of about 0.6 mm. 2 The above is an example of about 0.65 mm. 2 Above, for example, 0.7 mm 2 Above, for example, 0.75 mm 2 Above, for example, 0.85 mm 2 Above, for example, 0.95 mm 2 Above, for example, 1.00mm 2 Above, 1.1mm as an example 2 Above, for example, 1.2 mm 2 Above, 1.3mm as an example 2 Above, 1.4mm as an example 2 Above, 1.5mm as an example 2Above, 1.6mm as an example 2 Above, 1.7mm as an example 2 Above, 1.75mm as an example 2 Above, 1.8mm as an example 2 Above, 1.9mm as an example 2 The above is an example of a maximum of about 2 mm. 2 For example, in one embodiment, the radial cutout is about 0.85 mm. 2 ~about 2.5mm 2 , for example, about 1 mm 2 ~Approx. 2.25mm 2 , or about 1.25 mm 2 ~ approx. 2 mm 2 , or any range or value therebetween.

[0027] Stated differently, in one embodiment, the radial notch may have a cross-sectional area proportional to the cross-sectional area of ​​the proximal end of the protrusion, the distal end of the protrusion, or both. For example, the proximal end of the protrusion, the distal end of the protrusion, or both may define a cross-sectional area that includes the cross-sectional area defined by the radial notch. Thus, the radial cross-section of the radial notch may occupy about 5% or more, for example about 8% or more, for example 10% or more, for example 15% or more, for example 20% or more, for example 25% or more, up to about 30% or less, for example about 5% to about 30%, for example 8% to about 25%, or for example about 10% to about 20%, or any range or value therebetween, of the total cross-sectional area of ​​the proximal end of each protrusion. However, in one embodiment, the above range refers to the percent cross-sectional area occupied by the radial notch at the proximal end of the protrusion. As discussed above, the present disclosure has surprisingly discovered that when the radial notch has a radial cross-section sized in accordance with the above, the radial notch allows escape of gas bubbles from the sample without contributing to reoxygenation or causing overflow.

[0028] While the protrusions can have any radius, as discussed in more detail below, in one embodiment, the radial notch has a generally circular cross-sectional area. In such an embodiment, the radius of the radial notch has a length relative to the length of the radius of the protrusion (at the proximal end, the distal end, or both, as defined above), such as from about 5% to about 30%, for example from about 7.5% to about 27.5%, for example from 10% to about 25%, or any range or value therebetween.

[0029] Moreover, the radial notches may have any cross-sectional shape, such as circular, elliptical, square, triangular, nodular, or the like. However, in one embodiment, the radial notches may have a circular cross-section, such as circular or oval. Thus, in such an embodiment, and as will be discussed in more detail with respect to the following figures, the radial notches extend into the sidewall of each protrusion, along the entire length of each protrusion, from the distal end to the proximal end, without penetrating or piercing the sidewall of the protrusion. Stated differently, the radial notches form a portion of the sidewall of each protrusion, and do not extend to the inner portion of the protrusion. It should therefore be clear that any 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 the chamber or well that fits into the respective protrusion, and are not formed within the interior (e.g., hollow) of the protrusion.

[0030] In addition, as briefly mentioned, by using radial notches having the above sizes and dimensions, the radial notches allow the chamber or well to overflow and contain excess sample between the respective protrusion body and the sidewall of the sample chamber or well without contaminating the adjacent chamber(s) or well(s). Furthermore, as discussed in more detail below, in one embodiment, the cross-sectional area of ​​the protrusion (including the radial notch) may be larger at the proximal end than at the distal end, either alone or in combination with a well having a larger cross-sectional area at the proximal end than at the distal end. In such a manner, the total overflow storage volume may be increased without sacrificing headspace exclusion and evaporation prevention, for example, in one embodiment, the cross-sectional area of ​​the proximal end of the protrusion is larger than the cross-sectional area of ​​the distal end of the protrusion by 2% to about 15%, for example, about 3% to about 12%, for example, 5% to about 9%, or any range or value therebetween.

[0031] Furthermore, in one embodiment, the well may also have a larger cross-sectional area at the proximal end than at the distal end. In such a manner, the total overflow storage volume may be increased without sacrificing headspace exclusion and evaporation prevention, for example, in one embodiment, the cross-sectional area of ​​the proximal end of the protrusion is greater than the cross-sectional area of ​​the distal end of the protrusion by about 3% to about 16%, for example about 4% to about 12.5%, for example about 5% to about 10%, or any range or value therebetween. Furthermore, in one embodiment, the well may have a ratio of proximal end cross-sectional area to distal end cross-sectional area that is greater than the ratio of protrusion proximal end cross-sectional area to protrusion distal end cross-sectional area. In such an embodiment, both the well and the protrusion may have a taper as further described below, but at a different taper rate (with the well having a larger taper), resulting in additional storage volume and overflow prevention. Thus, the distal end of the protrusion, the proximal end of the protrusion, or both, are at least partially contacted by the sample or are completely immersed in the sample such that some of the sample is contained within a channel formed between the radial notch and the respective matching chamber or well sidewall, and a lesser amount is contained between the sidewall of the protrusion that does not include the radial notch and the respective chamber or well sidewall.

[0032] It should be noted that in one embodiment, each protrusion has only one notch (either for oxygen dissipation or for overflow in other applications). Specifically, in one embodiment, when shaped and sized according to the above, only a single radial notch is required to dissipate bubbles and provide an overflow contamination area. However, in further embodiments, one or more of the protrusions may include two notches, three notches, or four notches. In such embodiments, each notch may be shaped and sized as discussed herein. As discussed in more detail below, in one embodiment, at least one strut has two notches formed symmetrically across a centerline extending from the proximal end to the distal end of the strut.

[0033] In one such embodiment in which the protrusions include at least two notches, the distal end of the protrusions may include a spotting surface that is generally parallel to the bottom surface or the plane of the lid of the respective well. For example, the spotting surface may generally have the same cross-sectional shape as the respective protrusions and may have a diameter of about 0.5 mm to about 4.5 mm, for example about 1 mm to about 4 mm, for example about 1.5 mm to about 3.5 mm, or any value or range therebetween. Furthermore, in one embodiment in which one or more of the lid, protrusions, base, or wells are formed to have a non-clear color, the spotting surface may generally be transparent or matte.

[0034] However, it should also be understood based on the description of the method for determining oxygen consumption rate that the oxygen consumption rate is not determined based on visual inspection from above the sample, although the sample may in one embodiment completely surround or contact the distal ends of at least some or all of the protrusions. Instead, the oxygen consumption rate is determined in one embodiment utilizing a signal detector, such as a plate reader, that measures data emitted from the photoluminescent material, which is discussed in more detail below. Thus, in one embodiment, the substrate material used to form some or all of the protrusions, the lid, or the base including the chambers or wells corresponding to the protrusions, is formed from a semi-transparent material, which is distinguished from a transparent material by limiting the viewing of the sample through the material while still passing sufficient light to allow probe interrogation, as defined above. In particular, such a material can diffuse light while allowing the photoluminescent material to be measured according to the above method using a top-down plate reader, a bottom-up plate reader, or others as known in the art. In one embodiment, at least one of the base, lid, and one or more protrusions is translucent and may have a black, white, or matte appearance, or may be transparent.

[0035] Furthermore, the present disclosure has discovered that when the distal end of each longitudinal projection (e.g., the end of each projection opposite the end adjacent to the lid) is beveled or tilted 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 having a shorter length from the distal end to the lid) is adjacent to the radial notch, any air bubbles present in the sample are guided into the notch to facilitate removal of the air bubbles. Thus, in one embodiment, the distal end is beveled or tilted at an angle of about 5° or more, for example about 7.5°, for example about 10° or more, for example about 12.5° or more, for example about 15° or more, for example about 20° or more, for example 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 angled at an angle of about 3° to about 30°, for example about 3° to about 25°, for example about 5° to about 20°, or any range or value therebetween. In particular, when angles according to the above are used for the distal end that is beveled toward the radial notch, bubbles can be effectively removed from the sample while still allowing for sensitive oxygen consumption rate readouts at small sample sizes.

[0036] Moreover, as can be appreciated, in embodiments including one or more prongs with two or more notches, the prong(s) may also include two or more angled portions at the distal tip corresponding to the two or more prongs. For example, in an embodiment including two prongs, the angled distal tip may include two angled portions, each having a highest side adjacent a respective radial notch, and each having a distal-most point that meets approximately at the longitudinal centerline. In such a manner, bubbles may be effectively removed into the one or more notches for removal from the sample.

[0037] In addition, it has been found that when a lid is used having the projections described in the pending claims in combination with a beveled distal end, highly accurate height clearance (the distance between the distal most point of the distal end and the bottom surface of the chamber or well) is obtained. Due to such improved accuracy in height clearance, extremely low sample volumes may be used without loss of functionality, which may also contribute to improved sensitivity to measurements such as oxygen consumption rate, extracellular acidification, and other measurements without disturbing cell growth or bubble removal. Specifically, the beveled distal end allows for improved accuracy while excluding the headspace, and therefore oxygen, without contacting the bottom wall of the chamber or well (unlike oil-in-headspace solutions). Thus, the microtiter assembly according to the present disclosure may have an average height clearance between the distal-most point of each projection and the underside of the chamber or well when the lid clasp is not in contact with the base containing the multiple chambers of about 0.25 mm or less, for example about 0.20 mm or less, for example about 0.15 mm or less, for example about 0.1 mm or less, for example about 0.075 mm or less, for example about 0.05 mm or less, or any range or value therebetween. For example, in one embodiment, the microtiter assembly has an average height clearance of about 0.025 mm to about 0.25 mm, for example about 0.035 mm to about 0.15 mm, for example about 0.045 mm to about 0.1 mm, or any range or value therebetween.

[0038] Furthermore, since the lid and lid assembly including the protrusions described above allow for a highly consistent height clearance, in one embodiment, the height clearance can have a very low degree of variability, such that the variation in height clearance between adjacent protrusions / wells is about 10% or less, for example about 5% or less, for example about 2.5% or less, for example about 2% or less, for example about 1% or less, etc. In particular, the present disclosure has discovered that when microtiter assemblies are formed according to the above, increased sensitivity is further achieved due to reduced sample sizes as well as highly consistent height clearance, which further improves the sensitivity of the measurement.

[0039] For example, the lid according to the present disclosure may be used to measure oxygen consumption rates, extracellular acidification rates (ECARs), and others, as may be described herein, utilizing a fill volume of 100 μL or less, for example, about 90 μL or less, for example, about 80 μL or less, for example, about 70 μL or less, for example, about 60 μL or less, for example, about 60 μL or less, for example, about 50 μL or less, for example, about 40 μL or less, for example, about 30 μL or less, for example, about 25 μL or less, for example, about 20 μL or less, for example, about 15 μL or less, for example, about 10 μL or more, for example, about 15 μL or more, or any range or value therebetween. For example, in one embodiment, the fill volume may be about 10 μL to about 100 μL, for example, about 15 μL to about 90 μL, for example, about 20 μL to about 80 μL, or any range or value therebetween.

[0040] In other words, the lid according to the present disclosure allows lower seeding density to be used while maintaining measurable results.For example, in one embodiment, the lid assembly according to the present disclosure can be used with seeding density of about 100,000 cells or less, for example about 75,000 cells or less, for example about 50,000 cells or less, for example about 25,000 cells or less, for example about 12,500 cells or less, for example about 10,000 cells or less, for example about 7,500 cells or less, for example about 5,000 cells or less, for example about 2,500 cells or less, for example about 1,000 cells or more, for example 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, for example, from about 6,500 cells to about 90,000 cells, for example, from about 10,000 cells to about 75,000 cells, or any range or value therebetween.

[0041] Specifically, as noted above, by utilizing the assembly according to the present disclosure, oxygen ingress / back diffusion can be reduced while excluding the gaseous headspace from the well or chamber, while still accommodating small sample volumes. For example, oil-in-headspace methods that minimize oxygen contact require fill volumes of more than 100 μL to avoid the oil contacting the bottom of the well at the center of the meniscus, which would destroy the optical path and render the sample unmeasurable. Conversely, the assembly according to the present disclosure allows a more disk-shaped field of view to be formed by the contact of the sample and the respective protrusions. Thus, unlike prior art approaches, very small sample volumes can be used while still reducing oxygen ingress / back diffusion.

[0042] Thus, the assembly according to the present disclosure may benefit fluid samples containing various cell lines, for example, due to increased sensitivity and reduced oxygen intrusion / back diffusion. Specifically, as discussed above, the assembly according to the present disclosure may be used with adherent cell lines as known in the art, in addition to suspension cell lines. As understood in the art, suspension cell lines may be immobilized on coated plates (e.g., by centrifugation and the use of immobilizing coatings such as PDL, PLL, collagen, and fibronectin, as discussed in more detail below) and evaluated using any one or more of the methods described herein.

[0043] Furthermore, the assembly of the present disclosure can also be used with and benefit from 3D constructs.Examples of such constructs include spheroids fixed on coated plates (examples of which can be described in US Provisional Patent Application No. 63 / 276,099, the entirety of which is incorporated herein) and / or organoids, 3D culture systems that utilize scaffolds or matrix materials (e.g., electrospun fibrous scaffolds, collagen, hydrogel, and matrigel, including Lonza's RAFT™ 3D cell culture system), tissues and microtissues, and magnetic spheroids.

[0044] For example, one such magnetic spheroid method that can benefit from small sample size includes magnetic spheroid drive. As known in the art, magnetic spheroids can be measured or localized using various methods, for example, in combination with low-attachment plates, magnetic particles, and magnets (e.g., using polylysine, magnetic arrays, or the like). One example of a spheroid workflow includes, by way of example only, the Greiner workflow, in which cells are magnetized and formed into spheroids using a magnetic drive. After spheroid formation, the spheroids can be kept in the Greiner spheroid plate or transferred to another suitable microplate. Regardless of the plate selected, a magnetic drive can be used to localize the spheroids while inserting a lid according to the present disclosure to orient the spheroids to the base of the well. Furthermore, in one embodiment, the magnetic drive can be maintained during the assay to localize the spheroids.

[0045] Additionally or alternatively, as noted above, one or more of the protrusions may be hollow. Thus, in one embodiment, the hollow core of one or more of the protrusions may include a core magnet to aid in spheroid localization, allowing the lid to be placed without a magnetic drive. In one embodiment, one or more of the protrusions include a core magnet that is permanently affixed to the respective hollow. Conversely, in one embodiment, a lid assembly according to the present disclosure may include a protrusion overlay disposed between the protrusions and the cover block. The protrusion overlay includes one or more magnetic spines that extend into the hollow of one or more of the protrusions in a releasable manner and may be removed after spheroid localization or spheroid transport. In one such embodiment, the protrusion overlay may include a number of magnetic spines corresponding to the number of protrusions, such that when the protrusion overlay is placed, each protrusion hollow is at least partially occupied by a magnetic spine.

[0046] Moreover, it should be understood that other fluid samples can benefit from the lid according to the present disclosure, as can be understood by those skilled in the art.For example, the present disclosure can be used in conjunction with enzymes, isolated organelles, such as mitochondria, microorganisms, prokaryotes, eukaryotes, stem cells, cardiomyocytes, ex vivo samples embedded on microscope slides, and the like.Nevertheless, although examples of various cell lines and samples are provided, it should be understood that in one embodiment, any sample suitable for interrogation using a chamber or well according to the present disclosure that benefits from adapted sensitivity, reduced or controlled oxygen intrusion / back diffusion, reduced sample volume (such as increased cell-to-volume ratio), or combinations thereof, can be used according to the present disclosure.

[0047] Moreover, as can be understood 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, in spheroid assays, ultra-low attachment coatings, other common tissue culture coatings, or combinations thereof can be used. For example, ultra-low attachment coatings include BioFloat® Flex available from faCellitate, which helps to avoid non-specific surface binding of proteins and cells, and Lipidure® available from NOF Corporation. However, it should be clear that other culture coatings can be used as known in the art.

[0048] Nevertheless, the lid according to the present disclosure is in reversible contact with the base and is therefore not permanently fixed to the base. Specifically, each protrusion is in reversible association with a chamber or well. For example, in one embodiment, the lid clasp can maintain contact with the base during the desired test period using various known methods, such as gravity, weighted lid, or other. In one such embodiment, a cover is applied over the lid, which releasably attaches to the base and applies a downward force to the lid.

[0049] To achieve reversible attachment, the present disclosure has found that a certain wall clearance, defined as the distance between any portion of the sidewall of each protrusion and the sidewall of the chamber or well, is used to allow the lid to be removed while excess sample returns to the chamber or well. Specifically, if too little wall clearance is used, the lid will not be removable, but if the wall clearance is too large, air bubbles will be trapped between the portion of the sidewall that does not include the radial notch, reducing the ability of the air bubbles to escape completely through the notch. Thus, in one embodiment, the assembly according to the present disclosure has a wall clearance of about 0.9 mm or less, for example about 0.8 mm or less, for example about 0.7 mm or less, for example about 0.6 mm or less, for example about 0.5 mm or less, for example about 0.4 mm or less, for example about 0.3 mm or less, for example about 0.2 mm or less, for example about 0.1 mm or less, for example about 0.05 mm or less, for example about 0.025 mm or less, or any range or value therebetween.

[0050] In other words, the wall clearance must be significantly smaller than the clearance by the radial notch in order to allow the air bubbles to escape through the notch instead of being trapped between the sidewall of the protrusion and the sidewall of the corresponding well.Thus, in one embodiment, the radial notch defines a radial notch clearance defined as the distance between the point along the radial cross section having the maximum distance from the sidewall of each well (e.g., the apex of the radial notch) and the respective well sidewall, and the wall clearance is about 50% or less of the radial notch clearance, for example about 40% or less, for example about 30% or less, for example about 20% or less, for example about 10% or less, for example about 5% or less, for example about 2.5% or less, for example about 1% or less of the radial notch clearance.

[0051] Furthermore, since the lid assembly including the lid and the protrusions described above allows for a highly consistent wall clearance, in one embodiment, the wall clearance may have a very low degree of variability, such that the variation in wall clearance between adjacent protrusions / wells is about 10% or less, for example about 5% or less, for example about 2.5% or less, for example about 2% or less, for example about 1% or less. 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 may be permanently secured to the base once it is placed in conjunction with the base.

[0052] Regardless of the method used, a further advantage of the assembly of the present disclosure is that evaporation is limited due to small wall clearance while remaining reversible. Thus, unlike samples that utilize fixed or permanent lids and oil to remove headspace, samples according to the present disclosure can undergo further testing after cellular respiration measurements are completed. For example, the assembly of the present disclosure allows protein assays or fluorescence-based assays, such as post-assay immunofluorescence assays (including, for example, TOM20 and LC-3), to be completed after oxygen consumption rate testing.

[0053] Nonetheless, it should be understood that in addition to subsequent testing, additional simultaneous testing may be performed in conjunction with the fluorescence-based assay, also referred to as "multiplexing." Specifically, as discussed in further detail, the assemblies of the present disclosure are Society for Biomolecular Screening (SBS) compliant, and thus may be adapted to measure additional target analytes (H+, CO, CO2, CO ... 2 , O 2 In addition to the above, the present invention is compatible with existing signal detectors for measuring analytes (in addition to the above). Thus, it should be understood by those skilled in the art that any additional tests that do not interfere with the target analytes that may be multiplexed may be performed simultaneously, but in one embodiment, the additional tests may include stains (such as Hoechst and Calcein AM), fluorescent dyes, mitochondrial membrane potential (JC-1, e.g., TMRE / TMRM), reactive oxygen species (DHE), or combinations thereof.

[0054] Alternatively, multiplexing analytes may require the use of a separate signal detector that does not interfere with the target analyte(s) discussed herein. The additional signal detector 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 a combination thereof. Plate readers with these detection modes are commercially available. Alternatively, in one embodiment, the sensor may be interrogated from below the sensor, such as from below the lid of the device, and in such an arrangement, the signal must be able to pass through the wells and / or chambers so that it can be detected, for example, by including a transparent or semi-transparent material in the wells and / or chambers, or by passing or including one or more optical fibers through the lid and / or protrusions, such that the signal can be interrogated from above without the need for a transparent material on the distal end of the protrusion.

[0055] In addition, as briefly mentioned above and as can be understood by those skilled in the art, the substrate material forming the base, lid, protrusion, or combination thereof increases contact with the sample due to the ability to flood the chamber and the lack of an oil barrier between the sample and the headspace or lid. Thus, as can be apparent to those skilled in the art, the assembly of the present disclosure allows the sample volume and / or fill volume, substrate material, and optional low-oxygen permeable coating to be selected based on the cell line to be investigated. In particular, it is well known that different cells respire differently and therefore deoxygenate the sample at different rates. For example, in one embodiment, cells that breathe quickly may require a larger fill volume or may have low 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. In one embodiment, cellular respiration is measured independently of fill volume. When measuring oxygen depletion over a set time with appropriate sensitivity, it has an optimal oxygen consumption:fill volume ratio. If oxygen consumption is too high, oxygen depletion is too rapid to be measured, and if oxygen consumption is too low, oxygen depletion is not measurable at all. Identifying the range in which oxygen depletion can be detected with measurable sensitivity provides a method of measuring cellular respiration independent of fill volume. Nevertheless, in one embodiment, the material used to form or coat the substrate is a cellular respiration volume of about 600 cm, measured at 23° C. and 0% RH according to ASTM D 3985. 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 approx. 0cm 3 / m 2 / Has an oxygen permeability of less than or equal to 24 hours.

[0056] In addition, in one embodiment, when low oxygen back diffusion is required for sensitive samples, as discussed in more detail in the Examples below, the material used to form the substrate or to coat the substrate has a thickness of about 16 cm, measured at 23° C. and 0% RH according to ASTM D 3985. 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 permeability of about 0.01 cm 3 / m 2 / 24 hours ~ approx. 20cm 3 / m 2 / 24 hours, for example, approx. 0.05 cm 3 / m 2 / 24 hours ~ approx. 17.5cm 3 / m 2 / 24 hours, for example, approx. 0.1 cm 3 / m 2 / 24 hours - approx. 15cm3 / m 2 / 24 hours, or any range or value therebetween. Such low oxygen permeability can be useful for applications such as cancer research, as the necessary cell lines have low cell densities that are enabled by the use of the lid according to the present disclosure as discussed herein.

[0057] For example, in one embodiment, any part of the assembly can be formed from substrate materials having various forms and compositions, and can be derived from naturally occurring materials, naturally occurring materials that have been synthetically modified, or synthetic materials. Examples of suitable substrate materials include, but are not limited to, nitrocellulose, glass, silica, Teflon, metals (e.g., gold, platinum, and the like), and ceramics (including aluminum oxide, silicon oxide, and the like), composite materials, and laminates thereof. Suitable substrate materials include polymeric materials, e.g., polysaccharides such as agarose (e.g., available from Pharmacia under the trade names Sephadex® and Sephacyl®), polyacrylamides, polystyrenes, polyvinyl alcohols, copolymers of hydroxyethyl methacrylate and methyl methacrylate, polyesters including poly(ethylene terephthalate) and poly(butylene terephthalate); polyamides (such as nylon); polyethers including polyformaldehyde and poly(phenylene sulfide); polysaccharides such as those sold under the trademarks KAPTON (DuPont, Wilmington, Del.) and UPILEX (Ube polyimides, such as those manufactured by Epson Corporation (Head Office: Sanyo Denki Co., Ltd., Japan); polyolefin compounds, such as 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, cycloolefin polymers (COP), such as those sold under the trade names Zeonor® and Zeonex®, and cycloolefin copolymers (COC), such as those sold under the trade name Topas®, and mixtures thereof, and the like. Specific polymeric materials that may be used for the substrate material include organic polymers, either homopolymers or copolymers, naturally occurring or synthetic, crosslinked or non-crosslinked.A cover, which may be disposed over the lid, may also be formed from the same types of materials as provided herein for the base.

[0058] For example, in one embodiment, the substrate material for all or a portion of the assembly may be selected to be a material that has naturally low oxygen permeability, such as an acrylate polymer, which in one embodiment may be polymethylmethacrylate, alone or further coated with a polymer with ultra-low oxygen permeability. In one embodiment, any of the above substrate materials may be used, but at least a portion of the substrate material may be selected from silicon oxide, silicon nitride, silicon oxynitride, aluminum oxide, mixed oxides, flexible glass (Al alternating with polyacrylate layers on a PEN-based substrate, available as ClearProtect© 3D barrier coating from Antec), which have low oxygen permeability. 2 O 3 layers), ceramics and polymers, etc. 2 O 3 or SiO 2The lid is coated with a polymeric or ceramic compound such as a glass compound or any compound suitable for low temperature deposition processes such as atomic layer deposition (ALD), molecular vapor deposition, or plasma enhanced chemical vapor deposition (PECVD). Other surface treatments that may be commonly available include Actiplas, Aquacer, Carbocer, Lipocer, Plasmaclean, Silitec, or combinations thereof. Furthermore, in one embodiment, all of the sample contacting parts of the multiple protrusions are coated, alone or in combination with coating other parts of the assembly, in a material with ultra-low oxygen permeability. In particular, as further discussed with respect to the examples below, such coatings may further help prevent re-oxygenation of the sample after elimination of the head space. Furthermore, it should be understood by those skilled in the art that additional reagents may be stored in the lid and / or protrusions, either by lyophilizing or solubilizing the reagents in the reagent substrate, to further adjust the assay. Exemplary reagent substrates include D-Glucose-6-PO4, pyruvate, α-keto-glutarate, α-keto-butyrate, Ala-Gin, Sparker Malate Control, α-D-glucose, citric acid, succinic acid, D,L-β-hydroxy-butyrate, L-serine, acetyl-L-carnitine, γ-amino-butyrate, glycogen, D,L-α-glycerol-PO4, D,L,-isocitrate, fumaric acid, L-glutamic acid, L-ornithine, octanoyl-L-carnitine, α-keto-isocaproic acid, D-Glucse-1-PO4, L-lactic acid, cis-aconitic acid, L-malate, L-glutamine, tryptamine, palmitoyl-D,L-carnitine, L-leucine, and combinations thereof.Additionally, 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, nordihydro-guaiaretic acid, amitriptyline, meclizine, berberine, alexidine, phenformin, diclofenac, celastrol, trifuoperazine, papaverine, or combinations thereof. However, it should be understood that other reagent substrates, reagents, and inhibitors may be used as known in the art.

[0059] As discussed, in one embodiment, the assembly according to the present disclosure is directed to a microtiter well plate assembly. Thus, the plate cover and base may have 6, 8, 24, 96, 384, 1536, etc. corresponding projections / wells as known in the art. However, in one embodiment, the assembly of the present disclosure is particularly well suited for a microtiter plate assembly having 96 wells and corresponding projections.

[0060] Exemplary embodiments 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 in a manner that not only efficiently reads, but can also perform measurements faster than many conventional systems. However, in one embodiment, the probes and corresponding sensors detect H+, CO, CO 2 , O 2It should be understood that the device may be selected to be any device capable of sensing and reporting changes in the target analyte, such as a stimulatory device, a flow cytometer, a stimulatory system ... for measuring a flow rate of a target analyte, or a combination thereof.

[0061] For example, oxygen-sensitive photoluminescent probes capable of sensing and reporting the oxygen concentration of an environment in fluid communication with the probe are widely known, see, for example, U.S. Published Patent Applications 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 ... No. 862,770, No. 7,849,729, No. 7,749,768, No. 7,679,745, No. 7,674,626, No. 7,569,395 No. 7,534,615, No. 7,368,153, No. 7,138,270, No. 6,989,246, No. 6,689,438, No. 6,39 See, for example, Nos. 5,506, 6,379,969, 6,080,574, 5,885,843, 5,863,460, 5,718,842, 5,595,708, 5,567,598, 5,462,879, 5,407,892, 5,114,676, 5,094,959, 5,030,420, 4,965,087, 4,810,655, and 4,476,870, PCT International Published Application WO2008 / 146087, and European Published Patent Application EP 1 134 583, all of which are incorporated herein by reference. Such optical sensors are available from several sources, including Presens Precision Sensing, GmbH of Regensburg, Germany, Oxysense of Dallas, Texas, USA, and Agilent Technologies of Cork, Ireland.

[0062] 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 WO2012 / 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. These methods and techniques are suitable for use in determining the oxygen concentration in a test tube or well sealed by an apparatus according to the present invention.

[0063] However, while the use of phosphorescence-based sensors has been discussed thus far, it should be understood that optical sensors according to the present disclosure may utilize solid-state sensors, nanoparticle sensors, microparticle sensors, and / or magnetic sensors, or the like. For example, solid-state sensors may include one or more spots or films on a lid, base, protrusions, or combinations thereof, and particle-based sensors, such as sensors coated on beads, may generally be in solution, in suspension, embedded in a suitable polymer, or present in a sample container such as a well. Alternatively, in one embodiment, particle-based sensors may be added into cells or coated onto a surface or embedded in a suitable polymer. Nevertheless, such sensors may be used in a variety of applications, including optical, optical, and / or optical applications, as known in the art. 2 , pH, temperature, CO 2 Or a combination thereof. For example, in one embodiment, the sensor is a solid sensor coated on the distal end of the protrusion, a soluble sensor, a particulate sensor (e.g., bead, magnetic bead, such as present in a sample container such as a well plate), a planar sensor (e.g., a film or foil, such as on the lid and microplate), or the oxygen-sensitive phosphorescent probe is one or more sensor spots on the lid and microplate, or a combination thereof (e.g., a solid oxygen sensor on the protrusion and a soluble pH sensor in the medium).

[0064] Moreover, in one embodiment, the sensor can be an electrochemical or potentiometric sensor. Additionally or alternatively, electrodes can also be included in the well to measure electrical properties, including impedance. Regardless of the sensor selected, in one embodiment, and as discussed above, it should be understood that the well or chamber can also include one or more reference probes that generate a signal of known value for instrument calibration in the form of any of the sensors discussed above.

[0065] Regardless of the type of solid-state sensor selected, in one embodiment, merely by way of example, the sensor may be embedded in a permeable medium, such as a permeable medium selected from hydrogel, silicone, and matrigel. In some embodiments, the sensor is attached to at least one of the protrusions by solidifying or removing the medium (such as by drying, hardening, cooling, evaporation, or other techniques). The solid-state sensor may be applied by dipping or spotting the distal end of at least one of the protrusions into a mixture of the fluorescent indicator in the medium.

[0066] However, it should be understood that in certain embodiments, the sensor may be spotted or dipped onto all or a portion of one or more of the protrusions. It should further be understood that in certain embodiments, the sensor may be removably connectable to the body of one or more of the protrusions of the assembly. It should further be understood that in certain embodiments, the sensor may be integrally formed with one or more of the protrusions. Integral formation of the sensor on one or more of the protrusions may be accomplished by one or more techniques, such as vapor deposition, chemical coating, spin coating, dipping, and robotic spotting.

[0067] The assembly and process according to the exemplary embodiment of the present disclosure are well suited for measuring components in all different types of samples, such as biological samples. In one embodiment, for example, the system and process according to the exemplary embodiment of the present disclosure can be used to measure one or more components or parameters associated with components in cellular material. The one or more components can be contained in the medium surrounding the cell, or can be contained in the cell itself. In some embodiments, the biological sample to be tested can include cellular material derived from cells, such as organelles, mitochondria, or cell extracts. As a particular advantage, the measurement can be completed in a label-free manner.

[0068] 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, which in one embodiment has or is at risk of having a disorder. Nevertheless, in one embodiment, the cell sample can include primary cells, cells directly isolated or taken from living tissue or organ, cultured cells, and / or immortalized cells. For example, the cell sample can include primary cells, or cells directly isolated or taken from living tissue or organ, and then cultured in vitro. In one embodiment, the cell sample includes cells that are modified, such as cells from knockout mice or CRISPR KO libraries, that are genetically modified for heterotypic expression of a gene of interest and / or for suppressed expression of a gene. 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 in the medium. Moreover, as will be appreciated, in one embodiment, the cell sample comprises a plurality of cells, for example a plurality of cells described herein.

[0069] The cells to be tested may include any suitable cell sample, including, but not limited to, cultured cells, primary cells, human cells, nerves, 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, reptile cells, amphibian cells, and the like. The cells to be tested may also include three-dimensional cell samples, such as tissue samples, cell spheroids, biopsy samples, cell scaffolds, organ chips, and the like. Examples of parameters that may be measured and that are related to the above cell functions include carbon dioxide concentration, oxygen concentration or oxygen partial pressure, calcium ions, halogen ions, and the like. However, in one embodiment, the measured parameter is oxygen concentration, such as oxygen consumption. Through these tests, an understanding of what drives cell phenotype and function and / or the precise context of the cell environment or microenvironment can be obtained.

[0070] The assemblies and processes according to exemplary embodiments of the present disclosure may be used to measure metabolic data of living cells, or (micro)environmental conditions of any living cells. Cellular material to be tested may include, for example, bacterial cells, fungal cells, yeast cells, prokaryotic cells, eukaryotic cells, and the like. Cells that may be tested include mammalian cells, including animal cells and human cells. Specific cells that may be tested may include cancer cells, immune cells, immortal cells, primary cells, induced pluripotent stem cells, cells infected with viral or bacterial pathogens, and the like.

[0071] For example, in one embodiment, the assemblies and processes according to exemplary embodiments of the present disclosure can be used to aid in immunotherapy. Immunotherapy is a type of treatment that enhances a patient's immune system to fight cancer, infection, and other diseases. Immunotherapy processes can include adoptive cell-based therapy, such as the production of T cells, natural killer (NK) cells, monocytes, macrophages, combinations thereof, and the like. During T cell therapy, for example, 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 modified so that they have specific proteins called receptors. The receptors on the T cells are designed to identify and target unwanted cells in the body, such as cancer cells. The modified T cells are cultured in the 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 transplant therapy, one example of which is referred to as chimeric antigen receptor (CAR) T cell therapy. The use of T cells for adoptive T cell therapy or T cell transplantation therapy has recently increased due to great success in fighting blood diseases.In some embodiments, aspects of the present 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 present invention can be used to monitor T cell activation, T cell exhaustion, T cell metabolism including starting material and modified products, and the like.

[0072] NK cells are a type of cytotoxic lymphocyte that can seek out and destroy infected cells in the body. NK cells can exhibit very fast immune reaction responses. As a result, the use of NK cells in anti-cancer therapy has gained great interest and popularity. However, there are only limited numbers of NK cells in mammalian blood, which requires the NK cells to grow to a relatively high cell density in a bioreactor.

[0073] Cultivation of cells such as T cells, NK cells, or other mammalian cells typically requires some complicated process from inoculation to use in patients. The assembly and process of the present disclosure can be used to monitor cell metabolism during any point in the culture process to ensure that the cells are healthy and / or have the desired metabolic phenotype, and that the medium in which the cells are growing contains optimal levels of nutrients. The present system and process can be used, for example, to make adjustments to ensure metabolic compatibility of the cells as they are growing.

[0074] In addition to immune cells, the metabolism of cancer cells can also be monitored to provide an understanding of which nutrients fuel cancer cells. For example, the assemblies and processes according to exemplary embodiments of the present disclosure can reveal mechanisms or components that affect the metabolism of cancer cells to inhibit growth. The assemblies and processes according to exemplary embodiments of the present disclosure can also be used to determine the rate at which cancer cells can proliferate. 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 potential therapeutics that are mitochondrial responsible. The risk of mitochondrial toxicity can be evaluated, for example, with high specificity and sensitivity. In this manner, the mechanism of action of some mitochondrial poisons can be determined.

[0075] The systems and processes according to exemplary aspects of the present disclosure may also be used to help treat obesity, diabetes, and metabolic disorders by aiding in the discovery of relevant therapies. For example, the processes and systems may be used to measure the functional effects of genetic changes to metabolic pathway components. Nutrients used in healthy and diseased cell models may be investigated. Furthermore, fatty acid oxidation and glycolysis may be analyzed in different cell types, including stem cells.

[0076] Nevertheless, aspects of the present disclosure may be further understood in accordance with the following figure description.

[0077] 1A and 1B, embodiments of bases and lids for use in assemblies according to the present disclosure are illustrated. Specifically, as shown in FIG. 1A, a microtiter assembly 100 is illustrated having a base 102 having a plurality of wells 104 each having at least one sidewall 106 (more clearly shown in FIG. 1B) that defines a cavity 108. Additionally, FIG. 1A shows a lid 110 having a plurality of projections 112 extending longitudinally from the lid 110 generally perpendicularly from a proximal end 114 to a distal end 116. As more clearly shown in FIG. 1B, each projection 112 has a radial notch 122 extending from the proximal end 114 to the distal end 116 of each projection 112. Additionally, each projection 112 has an angled distal tip 124. In particular, as discussed above, the beveled distal tip 124 is angled relative to the plane of the well base 126 and / or lid 110 according to the ranges set forth above.

[0078] As shown in FIG. 1A, a first row 118 of wells 102 have respective cavities 108 that are at least partially occupied with corresponding protrusions 112. While FIG. 1A illustrates an assembly having 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 include a number of protrusions 112 extending throughout the entire assembly and corresponding to the number of wells 104 in the respective base 102. Furthermore, in one embodiment, each lid 110 can include one or more strips including protrusions 112, each strip can be attached at one or more ends to a frame (shown more clearly in FIG. 3D below). For example, with respect to FIG. 1A, eleven additional strips with corresponding protrusions 112 can be attached to the frame such that each well 104 is occupied by a corresponding protrusion 112 to form a complete 96 well microtiter plate 100.

[0079] 1B, lids 110 are shown separate from assembly 100 with projections 112 extending from a proximal end 114 to a distal end 116, each projection having a radial notch 122 extending into at least one sidewall 128 of each projection. As shown in FIGS. 1A and 1B, projections 112 are connected to supports 130. Moreover, although only a single row is shown in FIG. 1B, it should be understood that supports 130 may be used to connect multiple lids 110 to a frame (not shown in FIGS. 1A and 1B).

[0080] Similarly, Figures 2A-2F illustrate another embodiment similar to Figures 1A and 1B, where the lid 110 is in the form of a strip of eight protrusions 112. Figure 2A shows a bottom-up view of the lid 110. As shown in Figure 2A, the lid 100 in such an embodiment can have a width of about 12 mm or less, for example about 11 mm or less, for example about 10 mm or less, for example about 9 mm or less, for example about 5 mm or more, for example about 6 mm or more, for example about 7 mm or more, for example 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, for example about 6 mm to about 11 mm, for example about 7 mm to about 9 mm, or any range or value therebetween.

[0081] 2A, which more clearly shows the apex 151 of the radial notch, the lid 110 may have a length of about 100 mm or less, for example about 95 mm or less, for example about 90 mm or less, for example about 85 mm or less, for example about 80 mm or less, or for example about 60 mm or more, for example about 65 mm or more, for example about 70 mm or more, for example about 75 mm, 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, for example about 65 mm to about 90 mm, for example about 70 mm to about 85 mm, or any range or value therebetween.

[0082] Similarly, with reference to Figures 2B (viewed along cross section BB of Figure 2A) and 2C, in one embodiment, protrusion 112 has a height h1 from detent 150 to distal end 116, and a diameter d1, and lid 110 has a height h2 from the detent receiving portion (not shown / shown more clearly in Figure 4A) to well base 126, and a diameter d2. Using these dimensions, the volume of each well 104 and the volume of the corresponding protrusion 112 can be determined to determine the fluid volume displaced by lid 110. Furthermore, as shown in Figures 2B and 2C, heights h1 and / or h2 can be measured from the centerline 111 of each protrusion.

[0083] For example, in one embodiment, h1 is about 15 mm or less, for example about 14 mm or less, for example about 13 mm or less, for example about 12 mm or less, for example about 11.5 mm or less, for example about 7 mm or more, for example about 8 mm or more, for example about 9 mm or more, for example about 10 mm or more, for example 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, for example about 9 mm to about 13 mm, for example about 10 mm to about 12 mm, or any range or volume therebetween.

[0084] In addition, h2 may be about 17.5 mm or less, for example about 16.5 mm or less, for example about 15.5 mm or less, for example about 14.5 mm or less, for example about 13.5 mm or less, for example about 13 mm or less, for example about 8.5 mm or more, for example about 9.5 mm or more, for example about 10 mm or more, for example about 10.5 mm or more, for example about 11.5 mm or more, for example 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, for example about 10 mm to about 16.5 mm, for example about 11.5 mm to about 14.5 mm, or any range or volume therebetween. Furthermore, it should be understood that the ranges of h1 and / or h2 provided above may be measured from the center line 111 or the most distal point of the distal tip 127 as shown in FIG. 2B (most distal point 127) and FIG. 2C (center line 111).

[0085] Furthermore, in one embodiment, d1 may be 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 2 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, or any range or value therebetween. For example, in one embodiment, the inner diameter d1 may be about 1 mm to about 5 mm, for example, about 2 mm to about 4.9 mm, for example, about 3 mm to about 4.85 mm, or any value or range therebetween.

[0086] Similarly, in one embodiment, d2 may be about 7 mm or less, for example, about 35.5 mm or less, for example, about 30 mm or less, for example, about 25 mm or less, for example, about 20 mm or less, for example, about 15 mm or less, for example, about 10 mm or less, for example, about 7.5 mm or less, for example, about 5 mm or less, for example, about 5 mm or more, for example, about 5.5 mm or more, for example, about 6 mm or more, for example, about 6.3 mm or more, for example, about 6.4 mm or more, for example, about 6.45 mm or more. In one embodiment, the outer diameter d2 may be about 5 mm to about 35.5 mm, for example, about 5.5 mm to about 25 mm, for example, about 6 mm to about 20 mm, or any range or value therebetween. Furthermore, it should be understood that the ranges of d1 and / or d2 provided above may be measured at the widest or smallest diameter of each d1 and / or d2 in an embodiment in which the protrusion 112 is tapered.

[0087] In addition, as shown in FIG. 2B, in one embodiment, the protrusions 112 may taper toward the distal end 116 to provide further reduction in spillover without generally jeopardizing wall clearance and evaporation reduction. Thus, in one embodiment, one or more protrusions 112 have a centerline 111 that is generally perpendicular to the lid 110, and each protrusion has a taper 113 having an angle relative to the centerline 111 of about 2° or less, for example about 1.75° or less, for example about 1.5° or less, for example about 1.25° or less, for example about 1° or less, for example about 0.75° or less, or about 0.25° or more, for example about 0.4° or more, for example 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 about 0.25° to about 2°, for example, about 0.35° to about 1.75°, for example, about 0.55° to about 1.5°, or any range or value therebetween.

[0088] 2D and 2E, FIG. 2D is a top-down view of FIG. 2A, and FIG. 2E is a cross-sectional view taken along line DD. Holes 115 are illustrated adjacent each radial notch 122. Nevertheless, as shown most clearly in FIG. 2E, the beveled distal tip 124 is at an angle relative to the plane 119 of the lid 110, which may be generally planar as shown. Furthermore, although the planes 119 of the lid 110 and well base 126 were mentioned above as being in parallel planes, as shown in FIG. 2E, it should be understood that in one embodiment, the well base 126 is beveled at the same or similar angle as the beveled distal tip 124. In such a manner, the well base 126 has a consistent thickness (not shown) throughout its cross-section. However, as noted above, in one embodiment, the well base 126 can be in a plane that is generally parallel to the lid plane 119, such that the well base 126 has a thickness that increases toward the distal-most point of the beveled distal tip 124.

[0089] Finally, FIG. 2F illustrates a perspective view of the lid 110 of FIGS. 2A-2F in an embodiment having a hole 115, as shown, which may be adjacent to a radial notch 122.

[0090] Further, as shown in FIG. 3A, in one embodiment, the lid 110 can be a single piece having several projections 112 equal to the corresponding base 102 (not shown). Each projection 112 has a radial notch 122 extending into at least one sidewall 128 of each projection 112. In addition, the lid 110 includes a frame 132 having a flat portion 134 and a lip 136. The lip 136 can include one or more angled corners 138 or can have one or more right angle corners 140. In one embodiment, as shown in FIG. 3A and FIG. 3B, two of the corners can be angled to provide a reversible locking function to the lid 110 by providing a more snug fit and seal to maintain the lid 110 on the base (not shown) and to further aid in evaporation.

[0091] Furthermore, the lip 136 may also have a height h3 that maintains SBS compatibility but extends further along the base 102 than previous lids. For example, the height h3 in the y-direction may be about 10 mm or more, for example about 10.25 mm or more, for example about 10.5 mm or more, for example about 10.75 mm or more, for example about 11 mm or more, for example about 11.25 mm or more, for example about 11.5 mm or more, up to about 12 mm or less, for example about 11.75 mm or less, for example about 11.7 mm or less, for example about 10 mm to about 12 mm, or about 10.25 mm to about 11.75 mm, for example about 10.5 mm to about 11.7 mm, or any range or value therebetween. Specifically, the present disclosure has discovered that a lip 136 having a height h3 within the above range may further help reduce evaporation, which becomes more important when using reduced sample sizes.

[0092] 3B further illustrates the base 102 cooperating with the lid 110 of FIG. 3A. As shown, the base 102 includes a support 142 having a skirt 141 that includes a plurality of chambers or wells 104. In the illustrated embodiment, the base 102 also includes an angled corner 138 that releasably mates with the angled corner 138 of the lid of FIG. 3A. As discussed above, the lid 110 of FIG. 3A can cooperate with each well 104 of FIG. 3B 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 allow all of the gaseous headspace contained in each well 104 to escape such that each protrusion 112 contacts the sample (not shown) contained in the respective well 104.

[0093] Additionally, FIG. 3C illustrates an assembly 100 according to the present disclosure, in which the lid 110 (more clearly shown in FIG. 3A, as discussed above) is disposed on the base 102 (more clearly shown in FIG. 3B above) such that each protrusion 112 is in reversible association with a respective well 104 (more clearly shown in FIG. 3B).

[0094] 3D illustrates another embodiment of an assembly 100 according to the present disclosure, where the lid 110 is shown in two pieces, a frame 152 and a cover 153. Specifically, as shown, an array of strips 156 including protrusions 112 are releasably secured to the frame 152. In such a manner, the frame 152 can be shaped to include the correct number of strips 156 for the number of wells 104 included in each base 102. Moreover, in such an embodiment, the cover 153 and / or the frame 152 can be formed of any of the substrate materials discussed above, or alternatively, the frame 152 can be formed of a rigid material such as a metal, which in one embodiment can be aluminum. Nonetheless, in one such embodiment, the cover 153 can be clear or matte.

[0095] 4A and 4B, a microtiter plate assembly 200 is shown having a lid 210 having a planar portion 234 and a rim 236. As discussed above, in one embodiment, the frame (top 132) can be integral with the lid 210. Additionally, the lid 210 has a plurality of protrusions 212 extending from a proximal end 214 adjacent the lid 210 to a distal end 216 having an angled distal tip 224. As shown in FIG. 4A, the protrusions 212 can be generally hollow and can have an outer wall 246 that contacts the sample and an inner wall 248 located at an inner portion of each protrusion. However, it should be understood that even if the protrusions are hollow, the radial notches 222 do not penetrate through the outer wall 246 into the inner wall 248. In one embodiment, for example, the protrusions 212 are hollow because they are formed by injection molding, although it should be understood that any method known in the art, such as milling, can be used.

[0096] In addition, FIG. 4A more clearly shows the stop 250. As discussed above, the stop 250 can be used in conjunction with the radial notch 222 and beveled distal tip 224 to provide highly accurate height and wall clearance, which will be discussed in more detail with respect to FIG. 4B. Specifically, the stop 250 contacts a portion of the base 202 to position the projection 212 at a specific height to maintain the required height and wall clearance (shown in more detail in FIG. 4B). In one embodiment, the stop 250 can contact a continuous portion of the base 202, or alternatively, can contact a stop receiving portion 258. Nevertheless, as shown, the microtiter plate assembly 200 also includes a plurality of wells 244 that are configured to releasably contain the projections 212 in a reversible manner.

[0097] 2B, the projections 212 have a height h1 from the stop 250 to the distal end 216, and a diameter d1, and the lid 210 has a height h2 from the stop 250 height to the well base 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.

[0098] FIG. 4B includes a close-up depiction of the distal end 216 of the protrusion 212 of FIG. 3A. Specifically, as shown in FIG. 4B, the distal end 216 of the protrusion 212 has an angled distal tip 224 relative to the well base 226 (e.g., the underside of the interior of the well cavity). Moreover, FIG. 4B more clearly illustrates the sample 252, height clearance 254, wall clearance 256, radial notch 222, apex 251, and radial notch clearance 253, as discussed above. Specifically, as shown, a small but precise height clearance 254 (e.g., the distance between the distal-most portion of the protrusion 212 / the portion of the protrusion 212 closest to the well base 226) is close to the well base 226 to enable sensitive measurements without contacting the well base 226, which would disrupt cell growth and monolayer formation. Moreover, as shown, the wall clearance 256 is 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 .

[0099] Moreover, FIG. 4B shows 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 base 226). In such a manner, bubbles may be guided by the angle of the angled distal tip 224 relative to the radial notch 222, such that bubbles may be efficiently evacuated from the sample 252. Furthermore, as shown in FIG. 4B, the sample 252 has a height h that is slightly higher than the sum of the height of the angled portion of the distal tip 224 and the height clearance 254. In such a manner, the sample 252 may extend into the radial notch 222 such that overflow is prevented but headspace is completely eliminated.

[0100] 4C illustrates an embodiment of the present disclosure in which the protrusion 212 includes two radial notches 222 and two angled surfaces 225 on an angled distal tip 224 having a distal-most end adjacent the centerline C and an uppermost end adjacent the respective notch 222. Moreover, as shown, the protrusion 212 includes a spotting surface 260 that is approximately perpendicular to the well base 226.

[0101] 11A and 11B further illustrate additional embodiments of bases and lids for use in assemblies according to the present disclosure. Specifically, as shown in FIG. 11A, a microtiter assembly 100 is illustrated having a base 102 having a plurality of wells 104. Furthermore, FIG. 11A shows a lid 110 having a plurality of protrusions (not shown) extending longitudinally from the lid 110 as described above with respect to FIG. 1A. The lid 110 is shown viewed from above in FIG. 11B. In particular, the lid 110 can include a plurality of spacers 160 disposed on its outer surface 167. The spacers 160 can be located around a perimeter 166 of the lid 110 and / or at a center 168 of the lid 110, or any suitable location. The inventors have discovered that the use of such spacers 160 can prevent warping of the lid 110 by increasing the downward pressure on the base 102 via the lid 110 to provide more accurate test results and achieve sufficient sample volume consistency across the base 102 resulting in a more consistent oxygen depletion rate across the entire base 102.

[0102] 11B, multiple spacers 160 may be present in a first outer region 161 of the lid 110, multiple spacers 160 may be present in a second outer region 162 of the lid 110, and a single spacer 160 may be located in a central region 163 of the lid 110. However, it should be understood that additional spacers 160 may be located in any of the regions 160, 161, or 162 to achieve reduced warping of the lid 110. Additionally, the spacer 160 may be injection molded during formation of the lid 110 and may be formed from the same material as the remainder of the lid 110.

[0103] It should further be appreciated that the spacers 160 may have varying thicknesses in the y-direction to provide uniformity and reduced warping of the lid 110. However, in other embodiments, the spacers 160 may each have the same thickness. For example, in some embodiments, the spacers 160 may each have a thickness ranging from about 100 micrometers to about 500 micrometers, such as from about 125 micrometers to about 475 micrometers, such as from about 150 micrometers to about 450 micrometers.

[0104] In one particular embodiment, the spacers 160 in the central region 163 may have a thickness greater than the thickness of the spacers 160 in the first outer region 161 and the second outer region 162 to provide increased downward pressure in the center of the lid 110 where warping may occur more. In such an embodiment, the spacers 160 in the central region may have a thickness ranging from about 175 micrometers to about 500 micrometers, for example from about 200 micrometers to about 475 micrometers, for example from about 225 micrometers to about 450 micrometers, while the spacers 160 in the first outer region 161 and the second outer region 162 may have a thickness ranging from about 100 micrometers to about 250 micrometers, for example from about 125 micrometers to about 225 micrometers, for example from about 150 micrometers to about 200 micrometers.

[0105] As shown in FIG. 12, a comparison of the coefficient of variation of oxygen deficiency measurements in wells of the lid 110 of the microtiter plate assembly 100 of FIGS. 11A and 11B utilizing spacers 160 having various thicknesses compared to a lid 110 without a spacer indicates that the coefficient of variation (% CV) is reduced when a spacer 160 is utilized compared to when a spacer is not utilized.

[0106] Aspects of the present disclosure can be further understood by reference to the following non-limiting examples.

[0107] [Example 1] A lid according to the present disclosure was formed and positioned into each well by allowing the projections to sink into each well and then pressing the lid into place to allow controlled bubble dissipation. Oxygen intrusion was tested to indicate reoxygenation of fully deoxygenated samples by perfusing the samples with nitrogen gas and monitoring the deoxygenation level using OpTech by dOxyBeads® or Mocon or an oxygen-sensitive phosphorescent probe (MitoXpress® Xtra) and measuring oxygen intrusion using a fluorescent plate reader (CLARIOstar BMG Labtech, reader software reports probe emission intensity or lifetime as defined above). Plate preparations were performed in hypoxic bags flushed and filled with nitrogen gas to maintain sample deoxygenation. Negative (100% air saturation) and positive (0% O 2 ) control, as well as a sample containing an oil layer in the headspace.

[0108] As shown in Figure 5A, two samples with lid-seal / lid according to the present disclosure were tested, one with a radial notch filled with paraffin wax. As illustrated in Figure 5A, the lid with wax exhibited similar reoxygenation to the lid without wax, with a 1.75 mm 2 Figure 5B shows that radial notches with a radial cross section of 10 mm and a beveled distal end angled at 5° do not play a large role in oxygen back diffusion. Similarly, Figure 5B includes two samples prepared according to Figure 5A, except that the wells and bases contained an additional glass coating. Figure 5B illustrates that radial notches do not contribute significantly to oxygen back diffusion, even when the microplate wells were coated with a glass coating to reduce reoxygenation from the lid and well substrate.

[0109] Additionally, Figure 5C shows a comparison of oxygen ingress from both coated and uncoated microplate wells. As shown in Figure 5C, lids formed in accordance with the present disclosure exhibited low back diffusion rates of oxygen. Specifically, as shown in Figures 5A-5C, samples formed in accordance with the present disclosure effectively dissipated air bubbles and had comparable back diffusion to oil-in-headspace samples.

[0110] [Example 2] 1.75mm 2 A lid according to the present disclosure was formed having a radial cross section of 0.01 mm and a radial notch with a beveled distal end angled at 5°, 7.5°, and 10°. Each of the samples of the present invention was measured in two separate runs using A549 cells to test oxygen respiration. FIG. 6 illustrates oxygen consumption by A549 cells seeded at a density of 65,000 cells and a fill volume of 50 μL. As shown in FIG. 6, the lid formed according to the present disclosure was more sensitive to respiration, exhibiting a 5-fold increase in the rate of probe signal change compared to the oil-in-headspace sample, and also exhibited complete deoxygenation within the target time of 45 minutes (as shown, it was complete at 30 minutes, which is within the 45 minute target).

[0111] [Example 3] Samples according to the present disclosure were formed having the following dimensions:

[0112] [Table 1]

[0113] Examples according to the present disclosure were benchmarked against an oil-in-headspace control as well as an XF flux analyzer, as shown in Figure 7. As shown in Figure 7, the lid according to the present disclosure exhibited similar sensitivity to the Seahorse analyzer and much greater sensitivity compared to the oil-in-headspace samples, even at very low seeding densities.

[0114] [Example 4] Interrogation was performed in the same manner as in Example 1 above, except that a solid-state sensor of oxygen-sensitive photoluminescent dye contained in an oxygen-permeable carrier matrix was used, disposed at the beveled distal tip of the protrusion. Samples were interrogated using a plate reader (BioTek Cytation 5 from Agilent Technologies) as described above. Cell samples of 40,000 A549 cells per well were used in F12K medium. As shown in FIG. 8, the solid-state sensor disposed in the lid according to the present disclosure exhibited good oxygen depletion sensing over a 1-hour measurement with an average rate of 5.89 μs / h and a %CV of 12.82.

[0115] [Example 5] Interrogations were performed in the same manner as in Example 1, except that both changes in pH and oxygen depletion were measured. Measurements were performed on a BioTek Cytation 5 and a BMG Clariostar plate reader as discussed above. A pH calibration was obtained as shown in Figure 9, which illustrates compatibility with lids according to the present disclosure.

[0116] A cell sample of 30,000 A549 cells, XF DMEM medium with rotenone, antimycin A, and 5 mM HEPES was used in conjunction with the lid and process according to Example 1. Both pH and oxygen depletion were measured simultaneously within the sample. As shown in Figure 10A (pH) and Figure 10B (oxygen depletion), the lid of the present disclosure exhibited sensitive oxygen depletion measurements during multiplexing, as exemplified in this example with pH.

[0117] [Example 6] A cell sample of 30,000 A549 cells was plated overnight and then treated with tamoxifen for either 1 hour or 24 hours in the plate assembly of the present disclosure before the cells were evaluated to determine oxygen consumption levels and cell viability. Calcein AM and hoescht dye pre-assay were added to the cells. The cell culture medium was replaced with medium containing MitoXpress Xtra, and the cells were then imaged using a Cytation 5 with a GFP LED / Filter set. The MitoXpress Xtra assay was initiated by inserting the lid of the present disclosure onto the base of the plate assembly.

[0118] As shown in Figures 13-14, the ability to multiplex cell viability and mitochondrial respiration measurements is demonstrated, adding context to the data. It allows the user to determine if a compound or drug directly affects mitochondria, as mitochondrial inhibitors are revealed by a sharp decrease in oxygen consumption, or if the effect is due to broader cytotoxicity. Loss of viability is revealed by Calcein AM. As cells are treated over a 24-hour period, the assay is also used to determine the overall metabolic flexibility of the cells. If respiration is reduced but viability remains, the cells may be adapted to produce ATP by glycolysis in addition to OXPHO.

[0119] [Example 7] Cells were loaded with MitoTracker and hoechst as per manufacturer's instructions. Cells were washed with medium and then the medium was replaced with medium containing MitoXpress Xtra. Imaging was performed while the plate was incubated with compound using Cytation 5 with RFP LED / Filter set. The lid of the present disclosure was then placed on the base of the plate assembly and MitoXpress Assay was performed as normal.

[0120] FIG. 15 shows the effect of compounds on the mitochondrial network. Rotenone, antimycin, and oligomycin condensed and dissociated the network compared to the control. The effect on structure is then reflected in the rate of oxygen consumption, showing a direct correlation between structure and function. Different compounds have different effects; some promote fusion, others inhibit fusion, some cause fission, others limit fission. A novel insight in this method is that for the first time, changes in mitochondrial structure are correlated to functional changes in respiration in the same well as shown in FIG. 16.

[0121] [Example 8] Cells were added with 2.5μM JC-1 and hoechst for 45 minutes. Cells were washed three times in medium, then initial images were taken using Cytation 5 with GFP and RFP LED / Filter set. Medium was replaced with medium containing MitoXpress Xtra. Cells were treated with compound for 1 hour, then imaged as shown in Figure 17. Respiratory assay was started by inserting the lid of the present disclosure onto the base of the plate assembly. Cells were measured for oxygen consumption using MitoXpress Xtra filter cube and two monochromator measurements (Red 475ex, 535em, and Green 475ex 590em). JC-1 ratio was calculated by dividing red FI / green FI.

[0122] Simultaneous measurements of mitochondrial membrane potential (MMP) and oxygen consumption from the same well were demonstrated. Figures 17 and 18 show that the traditional method of measuring JC-1 using open well measurements is comparable when the lid is in the well. There is an improvement under the lid as the background signal is reduced. The fluorescent images in Figure 18 show JC-1 plus Hoechst staining before and after treatment in live cells. Figure 19 shows oxygen consumption traces and rate data from compound treated cells. Figure 20 shows kinetic JC-1 data reporting on MMP using PMT measurements of JC-1 ratios from the same wells as the MitoXpress Xtra data. Controls responded as expected. The assay detects changes in hyperpolarization, depolarization, and basal levels, as well as being able to relate changes in MMP to changes in mitochondrial respiration rate. The method can distinguish between loss of membrane potential due to uncoupling and inhibition of the electron transport chain, which would otherwise be undetectable.

[0123] [Example 9] Cells were counted and resuspended in medium with MitoXpress Xtra reagent. Cells were then plated onto PDL-coated microplates prior to a centrifugation step. Cells were treated with compounds before inserting the disclosed lid and starting measurements in a plate reader. Figure 21 shows that THP-1 cells grown in suspension respond to control compounds as expected. This validates that the method for attaching cells to PDL plates and MitoXpress® Xtra are suitable for measuring respiration.

[0124] [Example 10] Cells were loaded with 2.5 μM JC-1 and Hoechst for 45 min. Cells were washed 3 times in media and then initial images were acquired using Cytation 5 with GFP and RFP LED / Filter sets. JC-1 ratios shown in FIG. 23 were calculated by dividing red FI / green FI. Similar to the adherent cell assay, suspension cells were also loaded with JC-1 and imaged. Cells responded to the control compound as expected. As shown in FIG. 22A-D, it was possible to image cells after the MitoXpress Xtra assay without disturbing the monolayer.

[0125] These and other modifications and variations to the present disclosure may be practiced 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 substituted, both in whole or in part. Moreover, those skilled in the art should appreciate that the foregoing description is merely exemplary 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 comprising at least one well; a lid having at least one semi-transparent protrusion corresponding to at least one well; the at least one translucent protrusion extends from a proximal end adjacent the lid to a distal end having a radial notch and an angled tip, the angled tip having an angle of about 5° or greater with respect to a plane of the lid; A microtiter plate assembly, wherein a wall clearance is defined between a sidewall of the at least one protrusion and a sidewall of the at least one corresponding well, and a radial notch clearance is defined between an apex of the radial notch and the sidewall of the corresponding well, the wall clearance being less than or equal to 50% of the radial notch clearance.

2. 1. A microtiter plate assembly comprising: a base comprising a plurality of wells spaced in an array, each well adjacent to a second well; a lid comprising a plurality of translucent projections spaced in an array corresponding to the array of the plurality of wells, each projection adjacent a second projection; the plurality of translucent protrusions extend from a proximal end adjacent the lid to a distal end having a radial notch and an angled tip, the angled tip being at an angle of about 5° or greater relative to a plane of the lid; A microtiter plate assembly, wherein a height clearance is defined between the distal-most point of the distal end of each protrusion and a well base of a respective well, and the percent variation in the height clearance between the protrusion and the well and between the adjacent second protrusion and a second well is about 10% or less.

3. At least a portion of the assembly is about 600 cm ３ / m ２ and / or at least a portion of said assembly is formed from a substrate material having an oxygen transmission rate of about 120 cm ３ / m ２ 3. The microtiter plate assembly of claim 1 or claim 2, which is coated with a polymer or ceramic compound having an oxygen transmission rate of 24 hours or less.

4. At least a portion of the assembly is made of Al ２ O ３ , SiO ２ , silicon oxide, silicon nitride, silicon oxynitride, aluminum oxide, Al alternating with polyacrylate ２ O ３ 13. The microtiter plate assembly of claim 1 , wherein the microtiter plate assembly is coated with a polyimide or a combination thereof.

5. The radial notch is about 0.7 mm ２ and preferably said radial cutout is about 1.25 mm or more. ２ 2. A microtiter plate assembly according to any one of the preceding claims, having a radial cross section of:

6. 10. A microtiter plate assembly according to any one of the preceding claims, wherein the assembly is configured to measure oxygen consumption of a fluid sample having a volume of 100 μL or less.

7. 2. The microtiter plate assembly of claim 1, wherein the lid is in reversible association with the base.

8. 10. The microtiter plate assembly of any one of the preceding claims, wherein the radius of the radial notch has a length of about 5% to about 30% of the length of the radius of a cross-section of the protrusion including the radial notch at the proximal end of the protrusion, at the distal end of the protrusion, or at both the proximal and distal ends of the protrusion.

9. 10. The microtiter plate assembly of any one of the preceding claims, wherein the at least one protrusion has a proximal radius and a distal radius, the distal radius being about 2% to about 15% smaller than the proximal radius.

10. 10. The microtiter plate assembly of any one of the preceding claims, wherein the assembly is configured to measure oxygen depletion in samples having a seeding density of up to 250,000 cells for a period of up to 45 minutes.

11. 2. The microtiter plate assembly of claim 1, wherein the lid is milled, extruded, injection molded, or any combination thereof.

12. 10. The microtiter plate assembly of any one of the preceding claims, wherein the assembly comprises from about 1 to about 384 wells and from about 1 to about 384 corresponding projections.

13. 2. The microtiter plate assembly of claim 1, wherein the lid is a single piece.

14. 13. The microtiter plate assembly of claim 1, wherein the lid is formed from a frame and one or more rows including a plurality of translucent protrusions attached to the frame.

15. 15. The microtiter plate assembly of claim 14, wherein the lid includes eight rows, each row including twelve corresponding projections.

16. 10. The microtiter plate assembly of claim 1, wherein at least a portion of the assembly is formed from an acrylate polymer.

17. The microtiter plate assembly of any one of the preceding claims, wherein the lid comprises a plurality of spacers.

18. A method for measuring oxygen deficiency rate utilizing an assembly according to any one of claims 1 to 17, comprising the steps of: placing a sample and an oxygen sensitive probe into the at least one well; contacting the sample with the corresponding protrusion; measuring said oxygen depletion using a fluorescent plate reader.

19. 20. The method of claim 18, wherein the method further comprises measuring acidification.

20. 20. The method of claim 18 or 19, wherein the oxygen sensitive probe is selected from the group consisting of a solid sensor coated on the distal end of the protrusion, a soluble sensor, a particulate sensor, a planar sensor, one or more sensor points on the lid and microplate, or a combination thereof.

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

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