Headspace elimination type microtiter plate cover

Abstract

A microtiter plate assembly is disclosed. The microtiter plate assembly includes a base having a plurality of apertures and a lid having a plurality of protrusions corresponding to the plurality of apertures. Each protrusion contains a radial notch and an angled distal tip, providing a bubble-free seal, reduced oxygen back diffusion, and increased sensitivity even at small sample volumes.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0002] Test tubes and microtiter plates are often 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 so that it is not in fluid communication with the surrounding environment (usually achieved by providing an oil layer above the sample) and interrogating the oxygen-sensitive photoluminescent probe in the sample through the oil layer. Using an oil layer to seal the sample provides the additional benefit of the presence of a gaseous headspace confined between the sample and the oil layer. It is known that the gaseous headspace trapped below the oxygen barrier slows down the detection of changes in oxygen concentration because a relatively large supply of oxygen is available in such a gaseous headspace.

[0003] Although the oil layer is generally effective in sealing the sample from direct fluid communication with the surrounding environment and limiting the presence of the gaseous headspace below the oil layer, the oil layer is not a particularly effective oxygen barrier, is difficult to apply appropriately and consistently, and is labor intensive. However, the use of oil as a headspace eliminator requires an increase in sample volume in order to avoid a reduction in optical path length (e.g., this may be caused by the formation of a meniscus causing the oil to contact the bottom of the cell culture), which reduces sensitivity to a level below that required by many measurement devices. For example, such high sample volumes require correspondingly high seeding densities, making it difficult to study cells at sub-confluent densities, which is further exacerbated when studying cells that are metabolically altered at confluence. In addition, oil as a headspace eliminator also requires a step of applying the oil to each well, which makes contamination and error possible and eliminates the possibility of performing post-assay testing.

[0004] Other devices have been proposed, such as microtiter caps, that allow for increased visibility and control of sample spillage. However, none of the proposed devices are designed for metabolic interrogation purposes, as they neither restrict oxygen entry into the sample nor adequately dissipate gas bubbles from the sample when sealed.

[0005] Therefore, while devices have been proposed to eliminate gaseous headspace in test tubes and microtiter plates, there remains a need for an improved system and method suitable for measuring and monitoring oxygen consumption in which both headspace and bubble formation are reduced. Summary of the invention

[0006] The present disclosure generally relates to a microtiter plate assembly including a base and a cover. The base includes at least one hole, and the cover includes at least one translucent protrusion corresponding to the at least one hole. The at least one translucent protrusion extends from a proximal end adjacent to the cover to a distal end having a radial notch and an inclined tip, the inclined tip having an angle of about 5° or greater relative to a plane of the cover, and a wall gap is defined between a sidewall of the at least one protrusion and a sidewall of the at least one corresponding hole. In addition, a radial notch gap is defined between an apex of the radial notch and a sidewall of the corresponding hole, wherein the wall gap is 50% or less of the radial notch gap.

[0007] The present disclosure also generally relates to a microtiter plate assembly including a base and a cover. The base includes a plurality of holes spaced apart in an array, wherein each hole is adjacent to a second hole, and the cover includes a plurality of translucent protrusions spaced apart in an array corresponding to the array of the plurality of holes, wherein each protrusion is adjacent to a second protrusion. In addition, the plurality of translucent protrusions extend from a proximal end adjacent to the cover to a distal end having a radial notch and an inclined tip, wherein the inclined tip has an angle of about 5° or greater relative to the plane of the cover. In addition, a height gap is defined between the farthest point of the distal end of each protrusion and the base of the corresponding hole, wherein the percentage change in the height gap between the protrusion and the hole and the adjacent second protrusion and the second hole is about 10% or less.

[0008] In another aspect, at least a portion of the assembly comprises an oxygen permeability of about 600 cm 3 / m 2 / 24 hours or less, and / or at least a portion of the component is formed of a substrate material having an oxygen permeability of about 120 cm 3 / m 2 / 24 hours or less polymeric or ceramic composite coating. In one aspect, at least a portion of the component is coated with: Al2O3, SiO2, silicon oxide, silicon nitride, silicon oxynitride, aluminum oxide, Al2O3 alternating with polyacrylate, or a combination thereof.

[0009] In yet another aspect, the radial notch has a diameter of about 0.7 mm. 2 or greater radial cross-section, preferably wherein the radial recess has a radial cross-section of about 1.25 mm 2 In one aspect, 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, the length of the radius of the radial notch is about 5% to about 30% of the length of the radius of the cross-section of the protrusion including the radial notch. In yet another aspect, the at least one protrusion has a proximal radius and a distal radius, wherein the distal radius is about 2% to about 15% smaller than the proximal radius.

[0010] Furthermore, in one aspect, the assembly is configured to measure oxygen consumption of a fluid sample having a volume of 100 μL or less. In yet another aspect, the assembly is configured to measure oxygen consumption of a sample having a seeding density of 250,000 cells or less in 45 minutes or less.

[0011] On the one hand, the cover is reversibly associated with the base. On the other hand, the cover is milled, extruded, injection molded or a combination thereof. In addition, on the one hand, the assembly includes about 1 to about 384 holes and about 1 to about 384 corresponding protrusions. On the other hand, the cover is a single piece. Additionally or alternatively, on the one hand, the cover is formed by a frame and one or more rows containing a plurality of translucent protrusions attached to the frame. On the other hand, the cover contains eight rows, each containing twelve corresponding protrusions. In addition, on the one hand, at least a portion of the assembly is formed by an acrylate polymer. In addition, on the other hand, the cover may include a plurality of spacers.

[0012] The present disclosure also generally relates to a method for measuring oxygen consumption rate using an assembly according to any one or more of the aspects discussed above. The method includes placing a sample and an oxygen-sensitive phosphorescent probe into at least one well, contacting the sample with a corresponding protrusion, and measuring oxygen consumption with a fluorescence plate reader.

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

[0014] Other features and aspects of the disclosure are discussed in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The 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, in which:

[0016] Figure 1A An aspect of a microtiter plate assembly according to the present disclosure is shown;

[0017] Figure 1B Shown according to Figure 1A One side of the microtiter plate cover;

[0018] Figure 2A shows a top view of another aspect of a microtiter plate cover according to the present disclosure;

[0019] Figure 2B The cross section BB shows Figure 2A Microtiter plate cover;

[0020] Figure 2C Shows Figure 2A A side perspective view of a microtiter plate cover;

[0021] Figure 2D Shows Figure 2A Bottom view of a microtiter plate cover;

[0022] Figure 2E Along DD shows Figure 2D A cross-sectional view of

[0023] Figure 2F Shows Figure 2A A perspective view of a microtiter plate cover;

[0024] Figure 3A Another aspect of a microtiter plate cover according to the present disclosure is shown;

[0025] Figure 3B Shown with Figure 3A The microtiter plate cover corresponds to the microtiter plate base;

[0026] Figure 3C Shown with Figure 3B The microtiter plate base can be releasably associated with Figure 3A One side of the microtiter plate cover;

[0027] Figure 3D An aspect of a microtiter assembly according to the present disclosure is shown;

[0028] Figure 4A Another aspect of a microtiter plate assembly according to the present disclosure is shown;

[0029] Figure 4B yes Figure 4A an enlarged perspective view of the distal end of the protrusion;

[0030] Figure 4C is an enlarged perspective view of a distal end of a protrusion according to the present disclosure;

[0031] Figure 5A is a graph of reoxygenation over time according to Example 1;

[0032] Figure 5B is a graph of reoxygenation over time according to Example 1;

[0033] Figure 5C is a graph of reoxygenation over time according to Example 1;

[0034] Figure 6is a graph showing the change in oxygen consumption over time according to Example 2;

[0035] Figure 7 is a graph showing the change in oxygen consumption with cell seeding density according to Example 3;

[0036] Figure 8 is a graph of oxygen consumption versus time according to Example 4;

[0037] Fig. 9 is a graph of pH calibration according to Example 5;

[0038] Fig. 10A and Fig. 10B is a graph showing pH and oxygen consumption measured according to Example 5;

[0039] Fig.11A and Fig. 11B Another aspect of a microtiter plate assembly according to the present disclosure is shown;

[0040] Fig.12 The following figure shows the cover with spacers of different thicknesses compared to the cover without spacers. Fig. 11B Comparison of the coefficient of variation of oxygen consumption measurements in the wells of the lids of microtiter plate assemblies;

[0041] Fig.13 Images are shown to demonstrate cell viability of cells cultured in the plate assembly of the present disclosure after 1 hour and 24 hours for a control sample of culture medium and for culture medium containing tamoxifen;

[0042] Fig.14 is a graph showing oxygen consumption measured for cells grown in medium containing tamoxifen after 1 hour and 24 hours;

[0043] Fig.15 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;

[0044] Fig.16 is a graph showing oxygen consumption measured for cells grown in medium containing rotenone, antimycin and oligomycin compared to control (untreated) medium samples;

[0045] Fig.17 shows a comparison of images taken when using the cover of the plate assembly of the present disclosure compared to when not using the cover (open) when measuring JC-1;

[0046] Fig.18shows a comparison of images taken using a plate assembly of the present disclosure before and after treatment with FCCP and oligomycin;

[0047] Fig.19 is a graph showing oxygen consumption measured for cells grown in medium containing FCCP and oligomycin compared to cells grown in control (untreated) medium;

[0048] Fig. 20 is a graph showing the kinetic JC-1 ratio measured for cells grown in medium containing FCCP and oligomycin compared to cells grown in control (untreated) medium;

[0049] Fig.21 is the following figure, which shows the ability to measure the respiration of cells grown in suspension and treated with various compounds using the plate assembly of the present disclosure;

[0050] Figure 22A-22D are images showing that suspension cells cultured in a plate assembly of the present disclosure can be loaded with JC-1 and imaged; and

[0051] Fig.23 Shown for Figure 22A-22D JC-1 ratios calculated for cells imaged in Figure 5.

[0052] Repeat use of reference characters in the present specification and drawings is intended to represent the same or analogous features or elements of the invention. DETAILED DESCRIPTION Definition and test methods

[0053] Oxygen concentration can be determined or indirectly interrogated by placing oxygen-sensitive photoluminescent probes and fluid test samples within a plurality of wells in a microtiter plate, and determining the oxygen concentration within each well of the covered microtiter plate by exposing the oxygen-sensitive photoluminescent probes within each well to excitation radiation extending through protrusions extending therein or the bottom of the well to produce excited oxygen-sensitive photoluminescent material, measuring radiation emitted by the excited oxygen-sensitive photoluminescent material through the protrusions and the bottom of the well, and converting the measured emission to target analyte concentration based on known conversion algorithms, or measuring the probe / sensor signal in intensity or lifetime mode. In one aspect, a suitable photoluminescent probe for such measurement is Xtra, available from Agilent Technologies. Instruments suitable for reading oxygen-sensitive photoluminescent probes within cell samples are known and available from many sources, including the CLARIOstar plate reader from BMG Labtech GmbH in Ottenberg, Germany, and the Synergy HTX from BioTek, an Agilent Technologies company. However, the photoluminescent material may also include an indicator dye incorporated into an oxygen-permeable polymer matrix, and it should be understood that an electrochemical sensor may also be used to determine oxygen consumption. As used herein, the oxygen consumption rate (OCR) (also referred to as the oxygen consumption rate) may be calculated by sensing metabolites (O2) consumed in a culture medium sample, which may then be reported as a rate (analyte change over time); or by measuring or indirectly evaluating the analyte concentration at a preselected time point (end point). Conversely, the production of oxygen that results in an increase in oxygen concentration may also be determined by measuring the increase in oxygen over time.

[0054] The change in oxygen consumption can be determined in a sealed system or in an unsealed system. In one embodiment, the definition of OCR includes the following situations: oxygen consumption is not determined in a sealed system (e.g., a system that allows oxygen to diffuse back into the sample or a large amount of oxygen diffuses back into the sample); or oxygen consumption is oxygen consumption in the sample corrected for oxygen back diffusion into the sample; or oxygen consumption is oxygen consumption that is not corrected for oxygen back diffusion into the sample; or oxygen consumption is determined in a sealed system (e.g., a system that does not allow oxygen to diffuse back into the sample or a large amount of oxygen diffuses back into the sample); or oxygen consumption is equal to or substantially equal to oxygen consumption in the sample. In addition, in one embodiment, oxygen consumption is determined directly or indirectly, for example, inferred from an oxygen gradient measured (e.g., in a test well), or by measuring oxygen at a preselected time point.

[0055] As used herein, the extracellular acidification rate (ECAR) can be determined using a basal value or initial value of the proton efflux of a cell sample (e.g., a value based on a measurement of the proton efflux of a cell sample performed before forming a reaction mixture) and determining the proton efflux rate after forming a reaction mixture. For example, as used herein, (ECAR) can be calculated by sensing metabolites (H+) consumed or produced in a culture medium sample, which can then be reported in the form of a rate (analyte change over time); or measuring the analyte concentration at a preselected time point (end point). In one embodiment, changes in ECAR can be determined in a sealed system or a non-sealed system.

[0056] With respect to data processing, in one aspect, a signal (such as Signals can be output in relative fluorescence units (RFU) and can be processed to identify The non-linear oxygen response of the probe and any potential temperature equilibration were adjusted for by taking the natural logarithm of the RFU signal at each time point and subtracting the natural logarithm of the signal control. The oxygen calibration of the probe has an exponential fit, which makes it a simple way to linearize the signal. The level of temperature equilibrium and drift in the probe signal is captured in the signal control and explained by subtraction. This ensures that a larger portion of the oxygen consumption trace is linear, resulting in a more robust data analysis and reduced coefficient of variation (%CV) for the assay. This is especially important for higher rates in RFU or lifetime scales, where few measurement points fall within the linear range of the curve. The signal can be further processed by normalizing relative to the first reading of the assay to better evaluate and compare compound reactions.

[0057] As used herein, "translucence" or "translucent" means that the light transmittance or optical density of a material is within a range between transparent and opaque. In general, translucent materials allow light to pass diffusely. A translucent material will allow a higher level of electromagnetic radiation in the visible spectrum to pass than an opaque or substantially opaque material, but will allow a lower level of electromagnetic radiation in the visible spectrum to pass than a transparent or substantially transparent material. A translucent material can provide protection against phototoxicity while still allowing detection of cells. The result is that the cells cannot be observed from the top of the plate.

[0058] As used herein, when used to modify a value, the term "about", "approximately" or "generally" indicates that the value can be increased or decreased by 10%, such as 7.5%, 5%, such as 4%, such as 3%, such as 2%, such as 1%, and remain within the disclosed aspects. In addition, when used to describe the amount of a substance in a material, the term "substantially free" is not limited to being completely or completely free, and can correspond to the lack of any perceptible or detectable amount of the substance in the material. Thus, for example, when the amount of a substance in a material is less than the accuracy of an industrially acceptable instrument or test for measuring the amount of the substance in the material, the material is "substantially free" of the substance. In certain exemplary embodiments, when the amount of a substance in a 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, the material may be "substantially free" of the substance. Detailed description

[0059] Those skilled in the art will appreciate that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the present disclosure.

[0060] In general, the present disclosure relates to a microtiter assembly, the microtiter assembly comprising a cover having a plurality of protrusions and a base having a plurality of piston chambers (e.g., holes) corresponding to the respective protrusions, the microtiter assembly eliminating the headspace present in the existing microtiter assembly. Therefore, in this way, the headspace elimination type microtiter cover of the present disclosure can also improve oxygen back diffusion and entry, while allowing a reduction in sample amount and fill volume. The present disclosure unexpectedly found that the following microtiter plate assembly allows for reduction or elimination (including complete elimination) of ambient headspace oxygen, and also limits oxygen back diffusion (oxygen entry), while also allowing a reduction in sample amount, the microtiter plate assembly having a plurality of protrusions extending longitudinally from the cover, wherein each protrusion has a combination of a radial notch and a height gap within a specific range, and is combined with an inclined distal end.

[0061] In particular, in one aspect, the present disclosure has discovered that by utilizing a radial notch of a specific size extending in a longitudinal manner from the distal end to the proximal end of each protrusion, bubbles present in the sample chamber (e.g., a well) are evacuated while also preventing sample from escaping. In one aspect, the radial notch, which will be discussed in more detail below with respect to the accompanying drawings, has the following radial cross-section: approximately 0.575 mm 2 or larger, such as about 0.585mm 2 or larger, such as about 0.595mm 2 or larger, such as about 0.6mm 2 or larger, such as about 0.65mm 2 or larger, such as about 0.7mm 2 or larger, such as about 0.75mm 2 or larger, such as about 0.85mm 2 or larger, such as about 0.95mm 2 or larger, such as about 1.00mm 2 or larger, such as about 1.1mm 2 or larger, such as about 1.2mm 2 or larger, such as about 1.3mm 2 or larger, such as about 1.4mm 2 or larger, such as about 1.5mm 2 or larger, such as about 1.6mm 2 or larger, such as about 1.7mm 2 or larger, such as about 1.75mm 2 or larger, such as about 1.8mm 2 or larger, such as about 1.9mm 2 or larger, such as up to about 2mm2 For example, in one aspect, the radial cross-section of the radial notch is about 0.85 mm 2 To about 2.5mm 2 , such as about 1mm 2 To about 2.25mm 2 , or about 1.25mm 2 To about 2mm 2 , or any range or value therebetween.

[0062] In other words, on the one hand, the radial recess can have a cross-sectional area proportional to the cross-sectional area of ​​the proximal end of the projection, the distal end of the projection, or both. For example, the proximal end of the projection, the distal end of the projection, or both can define a cross-sectional area including the cross-sectional area defined by the radial recess. Therefore, the radial cross section of the radial recess can account for about 5% or more of the total cross-sectional area of ​​the proximal end of each projection, such as about 8% or more, such as about 10% or more, such as about 15% or more, such as about 20% or more, such as about 25% or more, at most about 30% or less, such as about 5% to about 30%, such as about 8% to about 25%, or such as about 10% to about 20%, or any range or value therebetween. However, on the one hand, the above-mentioned range refers to the cross-sectional area percentage of the radial recess at the proximal end of the projection. As discussed above, the present disclosure unexpectedly discovered that radial notches, when having a radial cross-section conforming to the above dimensions, can allow gas bubbles to escape from a sample without promoting reoxygenation or causing overflow.

[0063] Although the protrusion can have any radius, as will be discussed in further detail below, in one aspect, the radial recess has a substantially circular cross-sectional area. In such aspects, the length of the radius of the radial recess is about 5% to about 30%, such as about 7.5% to about 27.5%, such as about 10% to about 25%, or any range or value therebetween, relative to the length of the radius of the protrusion (at the proximal end, the distal end, or both as defined above).

[0064] In addition, the radial notch can have any cross-sectional shape, such as circular, elliptical, square, triangular, nodular, etc. However, in one aspect, the radial notch can have a circular cross-section, such as circular or elliptical. Therefore, in such an aspect, and as will be discussed in more detail below with respect to the accompanying drawings, the radial notch extends into the sidewall of each protrusion from the distal end to the proximal end along the entire length of the corresponding protrusion without penetrating or piercing the sidewall of the protrusion. In other words, the radial notch forms a portion of the sidewall of each protrusion and does not extend into the interior portion of the protrusion. Therefore, it should be clear that any notch or channel formed by the radial notch is formed between the portion of the sidewall formed by the radial notch and the sidewall of the conformal chamber or hole of the corresponding protrusion, and is not formed in the interior (e.g., cavity) of the protrusion.

[0065] In addition, as briefly mentioned, by using radial notches having the above-mentioned sizes and dimensions, the radial notches allow excess sample to be contained between the corresponding protrusion and the sidewall of the sample chamber or hole without overflowing the chamber or hole and contaminating one or more adjacent chambers or holes. In addition, as will be discussed in more detail below, in one aspect, 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 the cross-sectional area of ​​the hole being larger at the proximal end than at the distal end. In this way, the total overflow reservoir volume can be increased without sacrificing headspace elimination and evaporation prevention, for example, in one aspect, the cross-sectional area of ​​the proximal end of the protrusion is approximately 2% to about 15%, such as about 3% to about 12%, such as about 5% to about 9%, or any range or value therebetween than the cross-sectional area of ​​the distal end of the protrusion.

[0066] In addition, on the one hand, hole can also have a cross-sectional area larger at the proximal end than at the distal end. In this way, total overflow reservoir volume can be increased without sacrificing headspace elimination and evaporation prevention, for example, on the one hand, the cross-sectional area of ​​the proximal end of projection is about 3% to about 16% than the cross-sectional area of ​​the distal end of the projection, such as about 4% to about 12.5%, such as about 5% to about 10% or any scope or value therebetween. In addition, on the one hand, the ratio of the proximal cross-sectional area of ​​hole to the distal cross-sectional area can be greater than the ratio of the protrusion proximal cross-sectional area to the protrusion distal cross-sectional area. In such respects, both hole and projection can have a taper as further described below, but the taper percentage is different (the taper of the hole is larger), thereby providing more reservoir 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 or completely immersed in the sample, such that a portion of the sample is contained in the channel formed between the radial recess and the corresponding conformal chamber or hole side wall, and a smaller amount is contained between the side wall of the protrusion that does not contain the radial recess and the corresponding chamber or hole side wall.

[0067] It should be noted that, on the one hand, each protrusion has only one notch (whether for the dissipation of oxygen or for overflow for other purposes). That is, on the one hand, when formed and sized according to the above, only a single radial notch is needed to dissipate bubbles and provide an overflow containment area. However, on the other hand, one or more protrusions can include two notches, three notches, or four notches. In such aspects, each notch can be formed or sized as discussed herein. As will be discussed in more detail below, on the one hand, at least one strut has two notches, which are symmetrically formed across a centerline extending from the proximal end of the strut to the distal end.

[0068] In one such aspect in which the projection comprises at least two notches, the distal end of the projection may include a coating surface that is substantially parallel to the lower surface of the corresponding hole or the plane of the cover. For example, the coating surface may have a cross-sectional shape that is substantially the same as the corresponding projection, and a diameter of about 0.5 mm to about 4.5 mm, such as about 1 mm to about 4 mm, such as about 1.5 mm to about 3.5 mm, or any value or range therebetween. In addition, in one aspect in which one or more of the cover, projection, base, or hole are formed to have an opaque color, the coating surface may be substantially transparent or frosted.

[0069] However, although on the one hand, the sample can completely surround or contact the distal ends of at least some or all of the protrusions, it should also be understood based on the description of the method for determining the oxygen consumption rate that the oxygen consumption rate is not determined based on visual inspection from above the sample. Instead, the oxygen consumption rate is determined using a signal detector (such as on the one hand, a plate reader) that measures the data released from the photoluminescent material, which will be discussed in more detail below. Therefore, on the one hand, the substrate material used to form some or all of the bases of the multiple protrusions, covers, or bases containing chambers or holes corresponding to the multiple protrusions is formed by a translucent material, which is different from a transparent material in that observation of the sample through the material is limited, while still allowing enough light to pass through to enable probe interrogation, as defined above. In particular, such a material can diffuse light while allowing the photoluminescent material to be measured using a top-down plate reader, a bottom-up plate reader, or other plate readers known in the art according to the above method. In one aspect, at least one of the base, cover, and one or more protrusions can be translucent and have a black, white, or frosted appearance, or can be transparent.

[0070] In addition, the present disclosure has found that when the distal end of each longitudinal protrusion (e.g., the end of each protrusion opposite to the end adjacent to the cover) is canted / sloped at a specific angle relative to a plane parallel to the cover (or the bottom of the hole), so that the highest side of the distal end (e.g., the side of the distal end with a shorter length from the distal end to the cover) is adjacent to the radial notch, any bubbles present in the sample are directed to the notch to facilitate the removal of bubbles. Therefore, in one aspect, the distal end is canted / sloped at an angle of about 5° or greater, such as about 7.5°, such as about 10° or greater, such as about 12.5° or greater, such as about 15° or greater, such as about 20° or greater, such as about 25° or greater, up to about 30° or less relative to the cover or the bottom of the hole. For example, in one aspect, the distal end is canted / sloped at an angle of about 3° to about 30°, such as about 3° to about 25°, such as about 5° to about 20°, or any range or value therebetween. In particular, when angled according to the above, towards the distal end of the radial notch, air bubbles can be effectively removed from the sample while also allowing a highly sensitive reading of oxygen consumption rate at small sample sizes.

[0071] In addition, as can be appreciated, in aspects comprising one or more protrusions having two or more notches, the one or more protrusions can also contain two or more angled portions at the distal tip corresponding to the two or more protrusions. For example, in aspects comprising two protrusions, the inclined distal tip can include two angled portions, each angled portion having a highest side adjacent to a corresponding radial notch, and having and each having a farthest point that meets approximately at the longitudinal centerline. In this way, bubbles can be effectively removed to one or more notches for removal from the sample.

[0072] Furthermore, it has been found that when using a cover having a combination of a protrusion and an inclined distal end according to the pending claims, a very accurate height clearance (the distance between the farthest point of the distal end and the lower surface of the chamber or well) is obtained. Due to this improved accuracy of the height clearance, extremely low sample volumes can be used without loss of functionality, which can also help improve sensitivity to measurements such as oxygen consumption rate, extracellular acidification and other measurements without disrupting cell growth or bubble removal. That is, the inclined distal end allows for improved accuracy while eliminating headspace and therefore oxygen without contacting the lower wall of the chamber or well (unlike oil-in-headspace solutions). Therefore, when the stopper of the cover contacts the base containing multiple chambers, the average height gap between the farthest point of each protrusion and the lower surface of the chamber or hole according to the microtiter assembly of the present disclosure can be about 0.25 mm or less, such as about 0.20 mm or less, such as about 0.15 mm or less, such as about 0.1 mm or less, such as about 0.075 mm or less, such as about 0.05 mm or less, or any range or value therebetween. For example, in one aspect, the average height gap of the microtiter assembly is about 0.025 mm to about 0.25 mm, such as about 0.035 mm to about 0.15 mm, such as about 0.045 mm to about 0.1 mm, or any range or value therebetween.

[0073] In addition, because the cover assembly including the cover and the above-mentioned protrusions allows for a highly consistent height gap, in one aspect, the height gap can have a very low degree of variability, such that the variation in the height gap between adjacent protrusions / wells is about 10% or less, such as about 5% or less, such as about 2.5% or less, such as about 2% or less, such as about 1% or less. In particular, the present disclosure has found that when a microtiter assembly is formed according to the above, increased sensitivity is achieved even with reduced sample amounts, and the highly consistent height gap further improves the sensitivity of the measurement.

[0074] For example, the cap according to the present disclosure can be used to measure oxygen consumption rate, extracellular acidification rate (ECAR) and other measurements as can be described herein using the following filling volume: 100 μL or less, such as about 90 μL or less, such as about 80 μL or less, such as about 70 μL or less, such as about 60 μL or less, such as about 60 μL or less, such as about 50 μL or less, such as about 40 μL or less, such as about 30 μL or less, such as about 25 μL or less, such as about 20 μL or less, such as about 15 μL or less, such as about 10 μL or greater, such as about 15 μL or greater, or any range or value therebetween. For example, in one aspect, the filling volume can be from about 10 μL to about 100 μL, such as from about 15 μL to about 90 μL, such as from about 20 μL to about 80 μL, or any range or value therebetween.

[0075] In other words, the cap according to the present disclosure allows for the use of lower seeding densities while maintaining measurable results. For example, in one aspect, the cap assembly according to the present disclosure can utilize a seeding density of about 100,000 cells or less, such as about 75,000 cells or less, such as about 50,000 cells or less, such as about 25,000 cells or less, such as about 12,500 cells or less, such as about 10,000 cells or less, such as about 7,500 cells or less, such as about 5,000 cells or less, such as about 2,500 cells or less, such as about 1,000 cells or greater, such as about 2,500 cells or greater, or any value or range therebetween. For example, in one aspect, the seeding density is from about 2,500 cells to about 125,000 cells, such as from about 6,500 cells to about 90,000 cells, such as from about 10,000 cells to about 75,000 cells, or any range or value therebetween.

[0076] That is, as mentioned above, by utilizing an assembly according to the present disclosure, oxygen ingress / back diffusion can be reduced while eliminating gaseous headspace from the hole or chamber, while also being compatible with small sample volumes. For example, an oil headspace approach to minimize oxygen contact requires a fill volume greater than 100 μL to avoid oil contacting the center of the meniscus at the bottom of the hole, which would destroy the optical path and render the sample unmeasurable. In contrast, an assembly according to the present disclosure allows a more disc-shaped view to be formed by contact of the sample and the corresponding protrusion. Thus, unlike existing methods, very small sample volumes can be used while still reducing oxygen ingress / back diffusion.

[0077] Thus, for example, due to increased sensitivity and reduced oxygen ingress / back diffusion, the assembly according to the present disclosure can provide benefits for fluid samples containing a variety of cell lines. That is, as described above, in addition to suspension cell lines, the assembly according to the present disclosure can also be used with adherent cell lines known in the art. As understood in the art, suspension cell lines can be fixed on a coated plate (e.g., by using a centrifuge and fixing a coating such as, for example, PDL, PLL, collagen, and fibronectin, which will be discussed in more detail below) and assayed using any one or more of the methods described herein.

[0078] In addition, the components of the present disclosure may also be used with and provide benefits to 3D constructs. Examples of such constructs include spheroids (examples of which may be described in U.S. Provisional Patent Application No. 63 / 276,099, which is incorporated herein in its entirety) and / or organoids immobilized on coated plates, 3D culture systems utilizing scaffolds or matrix materials (e.g., electrospun fiber scaffolds, collagen, hydrogels, and artificial basement membranes, including Lonza's RAFT TM3D cell culture systems), tissues and microtissues, and magnetic spheroids.

[0079] For example, a magnetic spheroid method that can benefit from a small sample amount includes a magnetic spheroid drive. As known in the art, magnetic spheroids can be measured using a combination of various methods such as low adhesion plates, magnetic particles and magnets, or can be positioned (e.g., using polylysine, magnetic arrays, etc.). As an example only, an example of a spheroid workflow includes a Greiner workflow, in which cells are magnetized, and magnetic drive is utilized to form spheroids. After spheroid formation, the spheroid can be maintained in a Greiner spheroid plate or moved to another suitable microplate. Regardless of the selected plate, magnetic drive can be used to position the spheroid, while inserting a lid according to the present disclosure to orient the spheroid at the base of the hole. In addition, on the one hand, magnetic drive can be maintained during determination to position the spheroid.

[0080] Additionally or alternatively, as described above, one or more protrusions may be hollow. Thus, on the one hand, the hollow core of one or more protrusions may include a core magnet to assist in spheroid positioning, thereby allowing the cover to be placed without magnetic drive. On the one hand, one or more protrusions include a core magnet permanently attached to the corresponding cavity. Conversely, on the one hand, a cover assembly according to the present disclosure may include a protrusion cover arranged between the protrusion and the cover block. The protrusion cover includes one or more magnetic thorns that extend into the cavity of one or more protrusions in a releasable manner and can be removed after spheroid positioning or spheroid transportation. In one such aspect, the protrusion cover may include a number of magnetic thorns corresponding to the number of protrusions, so that when the protrusion cover is placed, each protrusion cavity is at least partially occupied by the magnetic thorns.

[0081] In addition, as will be appreciated by those skilled in the art, it will be appreciated that other fluid samples may benefit from a cover according to the present disclosure. For example, the present disclosure may 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, while examples of various cell lines and samples have been provided, it will be appreciated that, in one aspect, any sample suitable for interrogation using a chamber or well according to the present disclosure may be used according to the present disclosure, the sample benefiting from adjusted sensitivity, reduced or controlled oxygen entry / back diffusion, reduced sample volume (such as increased cell to volume ratio), or a combination thereof.

[0082] Additionally, as will be appreciated by those skilled in the art, one or more culture coatings may be used on the plates, protrusions, covers, or a combination thereof, based on the fluid sample selected. For example, as discussed above, coatings for suspension cell lines may include poly-D-lysine (PDL), poly-l-lysine (PLL), collagen, and fibronectin. Additionally, for spheroid assays, ultra-low attachment coatings, other common tissue culture coatings, or a combination thereof may be used. For example, suitable ultra-low attachment coatings include those available from faCellitate. Flex and available from NOF Corporation This helps to avoid non-specific surface binding of proteins and cells. However, it should be clear that other culture coatings known in the art may be used.

[0083] Nevertheless, the cover according to the present disclosure is reversibly in contact with the base and is therefore not permanently attached to the base. That is, each protrusion is reversibly associated with a corresponding chamber or hole. For example, in one aspect, the stopper of the cover can be kept in contact with the base using various known methods (such as gravity, weighted cover or other methods) during the desired test period. In one such aspect, a cover is applied to the cover, which is releasably attached to the base and applies a downward force to the cover.

[0084] In order to achieve reversible attachment, the present disclosure has found that using a specific wall gap (defined as the distance between any portion of the sidewall of each projection to the sidewall of the chamber or hole) enables the lid to be removed while returning excess sample to the chamber or hole. That is, if an overly small wall gap is used, the lid will not be removable, but if the wall gap is too large, bubbles are trapped between the portions of the sidewall that do not contain radial notches, thereby reducing the ability to completely discharge bubbles through the notches. Therefore, on the one hand, the wall gap of the assembly according to the present disclosure is about 0.9 mm or less, such as about 0.8 mm or less, such as about 0.7 mm or less, such as about 0.6 mm or less, such as about 0.5 mm or less, such as about 0.4 mm or less, such as about 0.3 mm or less, such as about 0.2 mm or less, such as about 0.1 mm or less, such as about 0.05 mm or less, such as about 0.025 mm or less, or any range or value therebetween.

[0085] In other words, the wall gap should be significantly smaller than the gap of the radial notch so that bubbles are expelled through the notch rather than being trapped between the sidewall of the protrusion and the sidewall of the corresponding hole. Thus, in one aspect, the radial notch defines a radial notch gap, which is defined as the distance between the point having the maximum distance from the sidewall of the corresponding hole along a radial cross section (e.g., the vertex of the radial notch) and the sidewall of the corresponding hole, wherein the wall gap is about 50% or less of the radial notch gap, such as about 40% or less of the radial notch gap, such as about 30% or less, such as about 20% or less, such as about 10% or less, such as about 5% or less, such as about 2.5% or less, such as about 1% or less.

[0086] Furthermore, because the cover assembly including the cover and the above-described protrusions allows for a highly consistent wall gap, in one aspect, the wall gap can have a very low degree of variability, such that the variation in wall gap between adjacent protrusions / apertures is about 10% or less, such as about 5% or less, such as about 2.5% or less, such as about 2% or less, such as about 1% or less. However, it should be understood that in one aspect, once placed in association with the base, the cover can be permanently attached to the base, such as aspects where the assembly is not intended to be reused.

[0087] Regardless of the method used, another advantage of the assembly of the present disclosure is that evaporation is limited due to the small wall gap while remaining reversible. Therefore, unlike samples with fixed or permanent caps and oil removal of headspace, samples according to the present disclosure can be further tested after cellular respiration measurements are completed. For example, the assembly of the present disclosure will enable protein assays or fluorescence-based assays such as post-assay immunofluorescence assays (including, for example, TOM 20 and LC-3) to be completed after oxygen consumption rate testing.

[0088] Nevertheless, it should also be understood that in addition to subsequent tests, additional simultaneous tests can be performed in conjunction with fluorescence-based assays, which can also be referred to as "multiplexing." That is, as will be discussed in more detail, since the components of the present disclosure are Society for Biomolecular Screening (SBS) compatible and are therefore compatible with existing signal detectors for measuring additional target analytes (including calcium, ATP, NADP / NADHP, temperature, mitochondrial membrane potential, reactive oxygen species, etc., in addition to H+, CO2, O2 discussed in more detail herein). Therefore, while it should be understood by those skilled in the art that any additional tests that do not interfere with the target analytes that can be multiplexed can be performed simultaneously, in one aspect, the additional tests can include stains (such as Hoechst and calcein AM), fluorescent dyes, mitochondrial membrane potential (JC-1, such as TMRE / TMRM), reactive oxygen species (DHE), or a combination thereof.

[0089] Alternatively, multiplexed analytes may need to use a separate signal detector that does not interfere with one or more target analytes discussed herein. Additional signal detectors may have one or more detection modes, such as absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, fluorescence polarization, imaging (including fluorescence imaging) or detection of a combination thereof. Plate readers with those detection modes are commercially available. Alternatively, on the one hand, the sensor can be interrogated from below the sensor, such as from below the cover of the device; in such an arrangement, the signal should be able to pass through the hole and / or chamber so that it can be detected, for example, by making the hole and / or chamber include a transparent material or a translucent material; or it can be interrogated from above without the need for a transparent material on the distal end of the protrusion, such as having one or more optical fibers passing through the cover and / or protrusion or contained in the cover and / or protrusion.

[0090] In addition, as briefly mentioned above, and as will be appreciated by those skilled in the art, due to the ability to overfill the chamber and the lack of an oil barrier between the sample and the headspace or lid, the substrate material forming the base, lid, protrusion, or a combination thereof, increases contact with the sample. Therefore, it may be clear to those skilled in the art that the components of the present disclosure allow the selection of sample volume and / or fill volume, substrate material, and optional low oxygen permeability coating based on the cell line to be studied. That is, it is well known that different cells breathe differently, and therefore samples can be deoxygenated at different rates. For example, on the one hand, fast-respiring cells may require a larger fill volume or may have a lower sensitivity to the substrate material or coating, while less active cell lines may benefit from a smaller sample volume and a coating or substrate material that limits the reverse diffusion of oxygen. On the one hand, the measurement of cellular respiration is independent of the fill volume. When oxygen consumption is measured within a set time with appropriate sensitivity, it has an optimal oxygen consumption: fill volume ratio. If the oxygen consumption is too high, the oxygen consumption is too fast to be measured; if the oxygen consumption is too low, the oxygen consumption cannot be measured at all. Identifying a range in which oxygen consumption can be detected with measurable sensitivity provides a method for measuring cellular respiration independent of fill volume. Nevertheless, in one aspect, the material used to form the substrate or coating the substrate has an oxygen permeability of about 600 cm2 as measured at 23°C and 0% RH according to ASTM D 3985. 3 / m 2 / 24 hours or less, such as about 300cm 3 / m 2 / 24 hours or less, such as about 150cm 3 / m 2 / 24 hours or less, such as about 120cm 3 / m 2 / 24 hours or less, such as about 100cm 3 / m 2 / 24 hours or less, such as about 80cm 3 / m 2 / 24 hours or less, such as about 50cm 3 / m 2 / 24 hours or less, such as about 25, cm 3 / m 2 / 24 hours or less, such as about 16cm 3 / m 2 / 24 hours or less, down to about 0cm 3 / m 2 / 24 hours or less or higher.

[0091] In addition, in one aspect, as will be discussed in more detail in the examples below, when sensitive samples require low oxygen back diffusion, the material used to form the substrate or coating the substrate has a relative humidity of about 16 cm as measured at 23° C. and 0% RH according to ASTM D 3985. 3 / m 2 / 24 hours or less low oxygen transmission rate, such as about 15cm 3 / m 2 / 24 hours or less, such as about 12.5cm 3 / m 2 / 24 hours or less, such as about 10cm 3 / m 2 / 24 hours or less, such as about 7.5cm 3 / m 2 / 24 hours or less, such as about 5cm 3 / m 2 / 24 hours or less, such as about 2.5cm 3 / m 2 / 24 hours or less, such as about 1cm 3 / m 2 / 24 hours or less, such as about 0.5cm 3 / m 2 / 24 hours or less, or any range or value therebetween. For example, in one aspect, the oxygen permeability of the material is about 0.01 cm 3 / m 2 / 24 hours to about 20cm 3 / m 2 / 24 hours, such as about 0.05cm 3 / m 2 / 24 hours to about 17.5cm 3 / m 2 / 24 hours, such as about 0.1cm 3 / m 2 / 24 hours to about 15cm 3 / m 2 / 24 hours, or any range or value therebetween. Such low oxygen permeability can be helpful in applications such as cancer research, as the necessary cell lines require low cell density, which can be achieved by using a cover according to the present disclosure as discussed herein.

[0092] For example, in one aspect, any portion of the assembly can be formed from a substrate material having a variety of 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, etc.) and ceramics (including alumina, silica, etc.), composite materials, and laminates thereof. Suitable substrate materials also include polymeric materials, including polysaccharides such as agar (e.g., can be commercially available from Pharmacia) and dextran (e.g., available under the trade name and commercially available, also from Pharmacia), polyacrylamide, polystyrene, polyvinyl alcohol, copolymers of hydroxyethyl methacrylate and methyl methacrylate, polyesters including poly(ethylene terephthalate) and poly(butylene terephthalate); polyamides such as nylon; polyethers including polyoxymethylene and poly(phenylene sulfide); polyimides such as those manufactured under the trademarks KAPTON (DuPont, Wilmington, Delaware) and UPILEX (Ube Industries, Ltd., Japan); polyolefin compounds including cycloolefin polymers, ABS polymers, Kel-F copolymers, poly(methyl methacrylate), poly(styrene-butadiene) copolymers, poly(tetrafluoroethylene), poly(ethylene vinyl acetate) copolymers, poly(N-vinyl carbazole), polytetrafluoroethylene, polypropylene, polystyrene, polycarbonates, cycloolefin polymers (COP) such as those manufactured under the trade name and ) and cyclic olefin copolymers (COC) (such as those sold under the trade name The invention relates to a polymer material that can be used for the substrate material, and ...

[0093] For example, in one aspect, the substrate material for all or a portion of the assembly can be selected to have a naturally low oxygen permeability, such as an acrylate polymer, which in one aspect can be polymethyl methacrylate, either alone or further coated with a polymer having an extremely low oxygen permeability. In one aspect, any of the above substrate materials can be used, but at least a portion of the substrate material is coated with a polymeric or ceramic composite material (such as a glass composite) having a low oxygen permeability or any composite material suitable for low temperature deposition processes (such as Al2O3 or SiO2), atomic layer deposition (ALD), molecular vapor deposition or plasma enhanced chemical vapor deposition (PECVD), such as ceramics and polymers, including silicon oxide, silicon nitride, silicon oxynitride, aluminum oxide, mixed oxides, flexible glass (Al2O3 layers alternating with polyacrylate layers on a PEN-based substrate can be 3D barrier coating from Antec). Other surface treatments that are commonly available include Actiplas, Aquacer, Carbocer, Lipocer, Plasmaclean, Silitec, or combinations thereof. In addition, in one aspect, all sample contacting portions of the plurality of protrusions are coated in a material having an extremely low oxygen permeability, either alone or in combination with other portions of the coating assembly. In particular, as will be further discussed with respect to the following examples, such coatings can further help prevent reoxygenation of the sample after elimination of the headspace. In addition, it will be appreciated by those skilled in the art that the reagents may be stored on the cap and / or protrusions by lyophilizing or dissolving the additional reagents in the reagent matrix to further adjust the assay. Exemplary reagent matrices include D-glucose-6-PO4, pyruvate, α-keto-glutaric acid, α-keto-butyric acid, Ala-Gin, Sparker MalateControl, α-D-glucose, citric acid, succinic acid, D,L-β-hydroxy-butyric acid, L-serine, acetyl-L-carnitine, γ-amino-butyric acid, glycogen, D,L-α-glycerol-PO4, D,L,-isocitric acid, fumaric acid, L-glutamic acid, L-ornithine, octanoyl-L-carnitine, α-keto-isocaproic acid, D-glucose-1-PO4, L-lactic acid, cis-aconitic acid, L-malic acid, L-glutamine, tryptamine, palmitoyl-D,L-carnitine, L-leucine, and combinations thereof. In addition, exemplary agents 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 myxothiazole, uncoupler 2,4-dinitrophenol, calcium, CaCl2, nordihydroguaiaretic acid, amitriptyline, meclizine, berberine, Alexidine, phenformin, diclofenac, triptolide, trifluoperazine, or a combination thereof. However, it should be understood that other reagent substrates, reagents and inhibitors as known in the art may be used.

[0094] As discussed, in one aspect, the assembly according to the present disclosure relates to a microtiter plate assembly. Thus, the plate cover and base may have 6, 8, 24, 96, 384, 1536 (and so on) corresponding protrusions / wells, as known in the art. However, in one aspect, the assembly of the present disclosure is particularly suitable for a microtiter plate assembly having 96 wells and corresponding protrusions.

[0095] Exemplary aspects of the present disclosure relate to components and methods for analyzing one or more biological components (including cellular parameters) contained in or associated with a sample of biological material (such as a cell culture). The methods and systems utilize light detection and ranging components in a manner that not only efficiently obtains readings, but also can make measurements faster than many conventional systems. However, it should be understood that in one aspect, the probe and corresponding sensor can be selected as any device capable of sensing and reporting changes in a target analyte (such as H+, CO, CO2, O2, or a combination thereof).

[0096] For example, oxygen-sensitive photoluminescent probes that can sense and report the oxygen concentration of an environment in fluid communication with the probe are well known. See, e.g., U.S. Published Patent Applications 2011 / 0136247, 2009 / 0029402, 2008 / 199360, 2008 / 190172, 2007 / 0042412, and 2004 / 0033575; U.S. Patents 8,242,162, 8,158,438, 7,862,770, 7,849,729, 7,749,768, 7,679,745, 7,674,626, 7,569,395, 7,534,615, 7,368,153, 7,13 and European Published Patent Application No. EP 1 134583, all of which are hereby incorporated by reference. Such optical sensors are available from a number of suppliers, including Presens Precision Sensing, GmbH, Regensburg, Germany, Oxysense, Dallas, Texas, USA, and Agilent Technologies, Cork, Ireland.

[0097] Methods and techniques for sensing oxygen within a test tube or well of a microtiter plate using an oxygen-sensitive photoluminescent probe are well known, as exemplified by WO 2012 / 052068, U.S. Patent Application Publication 2013 / 0280751, and U.S. Patent Application Publication 2014 / 0147882, all of which are incorporated herein by reference. These methods and techniques are applicable to determining the oxygen concentration within a test tube or well sealed with an apparatus according to the present invention.

[0098] However, although the use of phosphorescence-based sensors has been discussed so far, it should be understood that optical sensors according to the present disclosure may also utilize solid-state, nanoparticles, microparticles, and / or magnetic sensors, etc. For example, a solid-state sensor may include one or more points or membranes on a cover, base, protrusion, or a combination thereof, wherein a particle-based sensor (such as a sensor coated on a bead) may typically be in solution, in suspension, embedded in a suitable polymer, or located in a sample container such as a well. Alternatively, in one aspect, a particle-based sensor may be loaded into a cell or coated on a surface, or embedded in a suitable polymer. However, such sensors may include optics, O2, pH, temperature, CO2, or a combination thereof, as known in the art. For example, in one aspect, the sensor is a solid-state sensor coated on the distal end of the protrusion, a soluble sensor, a particulate sensor (which can be, for example, beads, magnetic beads, such as located in a sample container such as a well plate), a planar sensor (e.g., a membrane or foil, such as on a lid and a microplate), or where the oxygen-sensitive phosphorescent probe is one or more sensor spots on a lid and a microplate, or a combination thereof (e.g., a solid-state oxygen sensor on a protrusion and a soluble pH sensor in the medium).

[0099] Furthermore, in one aspect, the sensor may be an electrochemical sensor or a potentiometric sensor. Additionally or alternatively, electrodes may also be included in the wells to measure electrical characteristics including impedance. Despite the selection of sensors, in one aspect, and as described above, it should be understood that the wells or chambers may also contain one or more reference probes in the form of any of the above sensors that produce a signal of known value for instrument calibration.

[0100] Regardless of the type of solid sensor selected, in one aspect, by way of example only, the sensor can be embedded in a permeable medium, such as a permeable medium selected from a hydrogel, silicone, and an artificial basement membrane. In some aspects, the sensor is attached to at least one protrusion by curing or removing the medium, such as by drying, solidifying, cooling, evaporation, or other techniques. The solid-state sensor can be applied by dipping or coating the distal end of at least one protrusion in a mixture of a fluorescent indicator in the medium.

[0101] However, it should be understood that in some aspects, the sensor can be coated or dipped onto all or a portion of one or more protrusions. It should also be understood that in some aspects, the sensor can be removably connected to the body of one or more protrusions of the assembly. It should also be understood that in some aspects, the sensor can be integrally formed with one or more protrusions. The integral formation of the sensor on one or more protrusions can be achieved by one or more techniques (such as vapor deposition, chemical coating, spin coating, dip coating and robotic coating).

[0102] The components and methods according to the exemplary aspects of the present disclosure may be very suitable for measuring the composition in all different types of samples (such as biological samples). On the one hand, for example, the system and method according to the exemplary aspects of the present disclosure can be used to measure one or more components in a cell material or parameters related to the components in a cell material. The one or more components can be contained in the medium around the cell, or can be contained in the cell itself. In some embodiments, the biological sample tested can include cell materials derived from cells, such as organelles, mitochondria or cell extracts. Particularly advantageously, measurement can be completed in a label-free manner.

[0103] On the one hand, the cell sample is obtained from or derived from a subject, such as a human or non-human animal. On the one hand, the subject is a mouse, which suffers from or is at risk of developing a disorder on the one hand. Nevertheless, on the one hand, the cell sample may include primary cells, cells directly separated or harvested from living tissues or organs, cultured cells and / or immortalized cells. For example, the cell sample may include primary cells, or cells directly separated or harvested from living tissues or organs, and then cultured in vitro. On the one hand, the cell sample includes cells that have been modified (for example, genetically engineered for heterologous expression of target genes and / or genetically engineered for inhibiting gene expression), such as cells from knockout mice or CRISPR KO libraries. Nevertheless, on the one hand, the cell sample includes stem cells or cells derived from stem cells. Nevertheless, no matter what kind of cell is used, on the one hand, the cell sample includes culture medium, such as culture medium or growth medium, wherein the cell can be arranged in culture medium. In addition, as will be understood, on the one hand, the cell sample includes a variety of cells, such as a variety of cells described herein.

[0104] The cells tested may include any suitable cell samples, including but not limited to cultured cells, primary cells, human cells, neurons, 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, archaea cells, mammalian cells, bird cells, insect cells, reptile cells, amphibian cells, etc. The cells tested may also include three-dimensional cell samples, such as tissue samples, cell spheroids, biopsy samples, cell scaffolds, organ chips (organs-on-a-chip), etc. Examples of parameters that can be measured and related to the above cell functions include carbon dioxide concentration, oxygen concentration or oxygen partial pressure, calcium ions, hydrogen ions, etc. However, in one aspect, the measured parameter is oxygen concentration, such as oxygen consumption. Through these tests, people can understand what drives the cell phenotype and function and / or an accurate image of the cell environment or microenvironment.

[0105] The components and methods according to the exemplary aspects of the present disclosure can be used to measure the (micro) environmental conditions of living cell metabolism data or any living cell. For example, the cell material tested can include bacterial cells, fungal cells, yeast cells, prokaryotic cells, eukaryotic cells, etc. The cells that can be tested include mammalian cells, which include animal cells and human cells. The specific cells that can be tested include cancer cells, immune cells, immortal cells, primary cells, induced pluripotent stem cells, cells infected with viruses or bacterial pathogens, etc.

[0106] For example, on the one hand, the components and methods according to the exemplary aspects of the present disclosure can be used for adjuvant immunotherapy.Immunotherapy is a type of treatment that enhances the patient's immune system to fight cancer, infection and other diseases.For example, immunotherapy methods may include adoptive cell-based therapies, such as the production of T cells, natural killer (NK) cells, monocytes, macrophages, combinations thereof, etc.For example, during T cell therapy, T cells are taken out of the patient's blood.The T cells are then sent to a bioreactor for amplification or cultivation.In addition, T cells can be changed so that they have specific proteins called receptors.The receptors on T cells are designed to recognize and target unwanted cells in the body, such as cancer cells.The modified T cells are cultivated in a bioreactor to reach a certain cell density, and then supplied to the patient's body for fighting cancer or other diseases.T cell therapy may also be referred to as adoptive T cell therapy or T cell transfer therapy, an example of which is called chimeric antigen receptor (CAR) T cell therapy.Recently, the use of T cells for adoptive T cell therapy or T cell transfer therapy has been expanded due to its 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 transfer therapy. In some embodiments, aspects of the present invention can be used to monitor T cell activation, T cell exhaustion, T cell metabolism (including T cell metabolism of starting materials and modified products), etc.

[0107] NK cells are a type of cytotoxic lymphocyte that can seek out and destroy infected cells in the body. NK cells can exhibit very rapid immune response responses. Therefore, the use of NK cells in anti-cancer therapy has attracted great interest and popularity. However, there are only a limited number of NK cells in the blood of mammals, which requires NK cells to be grown to a relatively high cell density in bioreactors.

[0108] Cultivated cells (such as T cells, NK cells or other mammalian cells) generally require a somewhat complicated process from inoculation to use in patients. The components and methods 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 a desired metabolic phenotype, and that the culture medium in which the cells are grown contains optimized nutrient levels. For example, the system and method can be used to adjust to ensure the metabolic adaptability of the cells during growth.

[0109] In addition to immune cells, the metabolism of cancer cells can also be monitored to understand which nutrients provide nutrients for cancer cells. For example, the components and methods according to the exemplary aspects of the present disclosure can reveal the mechanism or components that affect the metabolism of cancer cells to inhibit growth. The components and methods according to the exemplary aspects of the present disclosure can also be used to determine the speed at which cancer cells can proliferate. The systems and methods of the present disclosure are also very suitable for use in toxicology. For example, the methods and components of the present disclosure can be used to detect mitochondrial liability in potential therapeutics. For example, the risk of mitochondrial toxicity can be assessed with high specificity and sensitivity. In this way, the mechanism of action of some mitochondrial poisons can be determined.

[0110] The systems and methods according to exemplary aspects of the present disclosure can also be used to assist in the treatment of obesity, diabetes, and metabolic disorders by helping to discover related therapies. For example, the methods and systems can be used to measure the functional effects of genetic changes on metabolic pathway components. Nutrients used in healthy and diseased cell models can be examined. In addition, fatty acid oxidation and glycolysis can be assessed in different cell types, including stem cells.

[0111] Nevertheless, various aspects of the present disclosure may be further understood from the following discussion of the drawings.

[0112] refer to Figure 1A and Figure 1B , showing one aspect of a base and a cover for use in an assembly according to the present disclosure. Figure 1A 1, a microtiter assembly 100 is shown having a base 102 having a plurality of holes 104 having at least one side wall 106 (in Figure 1B As more clearly shown in FIG. 1 , each side wall 106 defines a cavity 108. In addition, Figure 1A A cap 110 is shown having a plurality of protrusions 112 extending longitudinally from a proximal end 114 to a distal end 116 in a manner generally perpendicular to the cap 110. Figure 1BAs more clearly shown in FIG. 1 , each protrusion 112 has a radial notch 122 extending from the proximal end 114 to the distal end 116 of each protrusion 112. In addition, each protrusion 112 has an inclined distal tip 124. In particular, as described above, the inclined distal tip 124 is angled relative to the plane of the hole base 126 and / or the cover 110 according to the ranges discussed above.

[0113] like Figure 1A As shown, the first row 118 of holes 102 have corresponding cavities 108 that are at least partially occupied by corresponding protrusions 112. Figure 1A The assembly is shown with only a single row 118 of protrusions 112 attached to the cover 110, but it should be understood from the above that the cover 110 can extend across the entire assembly and include a number of protrusions 112 corresponding to the number of holes 104 in the corresponding base 102. In addition, in one aspect, each cover 110 can include one or more strips including protrusions 112, wherein each strip can be attached to the frame at one or more ends (see below). Figure 3D For example, regarding Figure 1A Eleven (11) additional strips having corresponding protrusions 112 may be attached to the frame such that each well 104 is occupied by a corresponding protrusion 112 to form a complete ninety-six (96) well microtiter plate 100 .

[0114] Next reference Figure 1B , the cover 110 is shown separated from the assembly 100, and has a protrusion 112 extending from a proximal end 114 to a distal end 116, each protrusion having a radial recess 122 extending into at least one side wall 128 of each protrusion. Figure 1A and Figure 1B As shown, the protrusion 112 is connected to the support member 130. Figure 1B Only a single row is shown in FIG. 1 , but it should be understood that the support member 130 can be used to connect multiple covers 110 to the frame ( Figure 1A and Figure 1B not shown).

[0115] Similarly, Figure 2A-2F Shows something like Figure 1A and Figure 1B In another aspect, the cover 110 is in the form of a strip of eight protrusions 112. Figure 2A A bottom view of the cover 110 is shown. Figure 2AAs shown, in such aspects, the width w of the cover 100 can be about 12 mm or less, such as about 11 mm or less, such as about 10 mm or less, such as about 9 mm or less, such as about 5 mm or more, such as about 6 mm or more, such as about 7 mm or more, such as about 8 mm or more, or any range or value therebetween. For example, in one aspect, the width w of the cover 110 is about 5 mm to about 12 mm, such as about 6 mm to about 11 mm, such as about 7 mm to about 9 mm, or any range or value therebetween.

[0116] In addition, if Figure 2A As shown, which more clearly shows the apex 151 of the radial notch, the length l of the cover 110 can be about 100mm or less, such as about 95mm or less, such as about 90mm or less, such as about 85mm or less, such as about 80mm or less, or such as about 60mm or more, such as about 65mm or more, such as about 70mm or more, such as about 75mm or more, or any range or value therebetween. For example, in one aspect, the length of the cover 110 is about 60mm to about 100mm, such as about 65mm to about 90mm, such as about 70mm to about 85mm, or any range or value therebetween.

[0117] Similarly, refer to Figure 2B (It is along Figure 2A cross-sectional view of BB) and Figure 2C In one aspect, the projection 112 has a height h1 and a diameter d1 from the stopper 150 to the distal end 116, and the cover 110 has a receiving portion (not shown / in FIG. Figure 4A 104 and the corresponding protrusion 112 to determine the volume of the fluid displaced by the cap 110. Figure 2B and Figure 2C As shown, height h1 and / or h2 may be measured from the centerline 111 of each protrusion.

[0118] For example, in one aspect, h1 is about 15 mm or less, such as about 14 mm or less, such as about 13 mm or less, such as about 12 mm or less, such as about 11.5 mm or less, such as about 7 mm or more, such as about 8 mm or more, such as about 9 mm or more, such as about 10 mm or more, such as about 11 mm or more, or any range or value therebetween. For example, in one aspect, the height h1 of the cover 110 is about 7 mm to about 15 mm, such as about 9 mm to about 13 mm, such as about 10 mm to about 12 mm, or any range or volume therebetween.

[0119] In addition, h2 can be about 17.5 mm or less, such as about 16.5 mm or less, such as about 15.5 mm or less, such as about 14.5 mm or less, such as about 13.5 mm or less, such as about 13 mm or less, such as about 8.5 mm or more, such as about 9.5 mm or more, such as about 10 mm or more, such as about 10.5 mm or more, such as about 11.5 mm or more, such as about 12 mm or more, or any range or value therebetween. For example, in one aspect, the height h2 of the cover 110 is about 8.5 mm to about 17.5 mm, such as about 10 mm to about 16.5 mm, such as about 11.5 mm to about 14.5 mm, or any range or volume therebetween. In addition, it should be understood that the ranges of h1 and / or h2 provided above can be measured from the farthest point of the centerline 111 or the distal tip 127, such as Figure 2B (farthest point 127) and Figure 2C (center line 111).

[0120] In addition, in one aspect, d1 can be about 5 mm or less, such as about 4.9 mm or less, such as about 4.8 mm or less, such as about 4.5 mm or less, such as about 4 mm or less, such as about 3.5 mm or less, such as about 3 mm or less, such as about 2.5 mm or less, such as about 2 mm or less, such as about 1.5 mm or less, such as about 1 mm or more, such as about 2 mm or more, such as about 3 mm or more, such as about 4 mm or more, such as about 4.5 mm or more, or any range or value therebetween. For example, in one aspect, the inner diameter d1 can be about 1 mm to about 5 mm, such as about 2 mm to about 4.9 mm, such as about 3 mm to about 4.85 mm, or any value or range therebetween.

[0121] Similarly, in one aspect, d2 can be about 7mm or less, such as about 35.5mm or less, such as about 30mm or less, such as about 25mm or less, such as about 20mm or less, such as about 15mm or less, such as about 10mm or less, such as about 7.5 or less, such as about 5mm or less, such as about 5mm or greater, such as about 5.5mm or greater, such as about 6mm or greater, such as about 6.3mm or greater, such as about 6.4mm or greater, such as about 6.45mm or greater. In one aspect, outer diameter d2 can be about 5mm to about 35.5mm, such as about 5.5mm to about 25mm, such as about 6mm to about 20mm, or any range or value therebetween. In addition, it should be understood that in the aspect that protrusion 112 is tapered, d1 and / or d2 provided above can be measured at the widest or smallest diameter of corresponding d1 and / or d2.

[0122] In addition, if Figure 2BAs shown, in one aspect, the protrusion 112 can taper toward the distal end 116 to provide further reduction in spillage without affecting the average wall gap and evaporation reduction. Therefore, in one aspect, one or more protrusions 112 have a centerline 111 that is substantially perpendicular to the cover 110, and each protrusion 112 has a taper 113, and the angle of the taper 113 relative to the centerline 111 is about 2° or less, such as about 1.75° or less, such as about 1.5° or less, such as about 1.25° or less, such as about 1° or less, such as about 0.75° or less, about 0.25° or greater, such as about 0.4° or greater, such as about 0.65° or greater, or any range or value therebetween. For example, in one aspect, the taper of one or more (or each) protrusion is about 0.25° to about 2°, such as about 0.35° to about 1.75°, such as about 0.55° to about 1.5°, or any range or value therebetween.

[0123] Next reference Figure 2D and Figure 2E ,in Figure 2D yes Figure 2A A top view of Figure 2E is a cross-sectional view taken along line DD. The aperture 115 is shown adjacent to each radial notch 122. Nevertheless, as Figure 2E As best shown in FIG. 1 , the angled distal tip 124 has an angle relative to the plane 119 of the cover 110, which can be substantially planar, as shown. Furthermore, although it is stated above that the plane 119 of the cover 110 is in a parallel plane with the hole base 126, as shown in FIG. Figure 2E , but it should be understood that in one aspect, the hole base 126 is inclined at the same or similar angle as the inclined distal tip 124. In this way, the hole base 126 has a consistent thickness (not shown) across its entire cross-section. However, as described above, in one aspect, the hole base 126 can be in a plane that is generally parallel to the plane 119 of the cap, such that the thickness of the hole base 126 increases toward the farthest point of the inclined distal tip 124.

[0124] at last, Figure 2F Shows Figure 2A-2F 1. A perspective view of the cover 110 of FIG. 1 is shown, in aspects having an aperture 115, the aperture 115 may be adjacent to the radial notch 122. As shown in FIG.

[0125] In addition, if Figure 3AAs shown, in one aspect, the cover 110 can be a single piece having a plurality of protrusions 112 equal to the corresponding base 102 (not shown). Each protrusion 112 has a radial notch 122 extending into at least one side wall 128 of each protrusion 112. In addition, the cover 110 includes a frame 132 having a planar portion 134 and a lip 136. The lip 136 can include one or more angled corners 138, or can have one or more square corners 140. In one aspect, as shown, the cover 110 can be a single piece having a plurality of protrusions 112 equal to the corresponding base 102 (not shown). Each protrusion 112 has a radial notch 122 extending into at least one side wall 128 of each protrusion 112. In addition, the cover 110 includes a frame 132 having a planar portion 134 and a lip 136. The lip 136 can include one or more angled corners 138, or can have one or more square corners 140. Figure 3A and Figure 3B As shown, two of the corners may be angled to provide a reversible locking function to the cover 110 by providing a tighter fit and seal to retain the cover 110 on the base (not shown) and further aid in evaporation.

[0126] In addition, the lip 136 can also have a height h3 that maintains SBS compatibility but extends further along the base 102 beyond the previous cover. For example, the height h3 can be about 10 mm or more, such as about 10.25 mm or more, such as about 10.5 mm or more, such as about 10.75 mm or more, such as about 11 mm or more, such as about 11.25 mm or more, such as about 11.5 mm or more, up to about 12 mm or less, such as about 11.75 mm or less, such as about 11.7 mm or less, such as about 10 mm to about 12 mm, or about 10.25 mm to about 11.75 mm, such as about 10.5 mm to about 11.7 mm, or any range or value therebetween. That is, the present disclosure has found that a lip 136 having a height h3 within the ranges discussed above can further help reduce evaporation, which is of greater importance when using reduced sample amounts.

[0127] also, Figure 3B Shown with Figure 3A As shown, the base 102 includes a support member 142 having a skirt 141 that accommodates a plurality of chambers or holes 104. In the illustrated aspect, the base 102 also includes an angled corner 138 that is aligned with the base 102. Figure 3A The angled corners 138 of the cover are releasably associated. As discussed above, Figure 3A The cover 110 can be connected with Figure 3B Each hole 104 of the cover 110 is fitted to occupy a portion of the cavity 108 formed by the hole sidewall 108. Although not shown, it should be understood that when a sample is present, the cover 110 can displace some or all of the gas located between the cover 108 and the sample (not shown), and in one aspect, can exhaust all of the gaseous headspace contained in each hole 104 so that each protrusion 112 contacts the sample (not shown) contained in the corresponding hole 104.

[0128] also, Figure 3C An assembly 100 according to the present disclosure is shown, wherein a cover 110 (as discussed above) is Figure 3A ) is arranged on the base 102 (on the top Figure 3B ), so that each protrusion 112 is reversibly associated with a corresponding hole 104 (in Figure 3B more clearly shown in ).

[0129] Figure 3D Another aspect of an assembly 100 according to the present disclosure is shown, wherein the cover 110 is shown in two parts, a frame 152 and a cover 153. That is, as shown, a plurality of strips 156 containing rows of protrusions 112 are releasably attached to the frame 152. In this manner, the frame 152 can be formatted to include the correct number of strips 156 for the number of holes 104 contained in the corresponding base 102. Further, in such an aspect, the cover 153 and / or the frame 152 can be formed from any of the base materials discussed above, or alternatively, the frame 152 can be formed from a rigid material such as a metal, which can be aluminum in one aspect. Nevertheless, in one such aspect, the cover 153 can be transparent or frosted.

[0130] Next reference Figure 4A and Figure 4B , shows a microtiter plate assembly 200 having a cover 210 having a planar portion 234 and a lip 236. As discussed above, in one aspect, the frame (132 above) can be integral with the cover 210. In addition, the cover 210 has a plurality of protrusions 212 extending from a proximal end 214 adjacent the cover 210 to a distal end 216 having an inclined distal tip 224. Figure 4A As shown, the protrusions 212 can be generally hollow and have an outer sidewall 246 that contacts the sample and an inner sidewall 248 located on the inner portion of each protrusion. However, even though the protrusions are hollow, it should be understood that the radial notches 222 do not penetrate through the outer sidewall 246 into the inner sidewall 248. In one aspect, for example, the protrusions 212 are hollow because they are formed by injection molding, however, it should be understood that any method known in the art, such as milling, may be used.

[0131] also, Figure 4A More clearly shown is the stop block 250. As discussed above, the stop block 250 can be used in conjunction with the radial notch 222 and the angled distal tip 224 to provide highly precise height and wall clearance, which will Figure 4B That is, the stopper 250 contacts a portion of the base 202 to arrange the protrusion 212 at a specific height to maintain the necessary height and wall clearance (at Figure 4B244). In one aspect, the stopper 250 can contact a continuous portion of the base 202, or alternatively, can contact the stopper receiving portion 258. Nevertheless, as shown, the microtiter plate assembly 200 also includes a plurality of holes 244 that are configured to reversibly releasably receive the protrusions 212.

[0132] In addition, if Figure 4A As more clearly shown in the above Figure 2B As discussed, the protrusion 212 has a height h1 and a diameter d1 from the stopper 250 to the distal end 216, and the cap 210 has a height h2 and a diameter d2 from the height of the stopper 250 to the hole base 226. Using these dimensions, the volume of the corresponding hole 204 and the volume of the corresponding protrusion 212 can be determined to determine the volume of fluid displaced by the cap 210.

[0133] Figure 4B Contains Figure 3A A close-up depiction of the distal end 216 of the protrusion 212. Figure 4B As shown, the distal end 216 of the protrusion 212 has a distal tip 224 that is inclined relative to the hole base 226 (e.g., the lower surface inside the hole cavity). In addition, Figure 4B The sample 252, height gap 254, wall gap 256, radial notch 222, apex 251, and radial notch gap 253 are more clearly shown, as discussed above. That is, as shown, the small but precise height gap 254 (e.g., the distance between the most distal 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 allow sensitive measurements without touching the well base 226 (which would interrupt cell growth and monolayer formation). In addition, as shown, the wall gap 256 is very small so that the protrusion 212 can be removed from the well 244 while still directing any bubbles to the radial notch 222.

[0134] also, Figure 4B The radial notch 222 is shown positioned adjacent to the highest portion of the inclined distal tip 224 (e.g., the portion of the distal end 216 that has the shortest distance from the proximal end 224 or the portion of the inclined distal tip 224 that is farthest from the well base 226). In this way, bubbles can be directed to the radial notch 222 by the angle of the inclined distal tip 224, so that bubbles can be effectively expelled from the sample 252. In addition, as shown in FIG. Figure 4B As shown, the sample 252 has a height h that is slightly higher than the sum of the height of the inclined portion of the distal tip 224 and the height gap 254. In this way, the sample 252 can extend into the radial recess 222 so that overflow is prevented, but the headspace is completely eliminated.

[0135] in addition, Figure 4C An aspect of the present disclosure is shown wherein a protrusion 212 includes two radial notches 222 and two angled surfaces 225 on an inclined distal tip 224, the angled surfaces 225 having a distal-most end adjacent the centerline C and a highest end adjacent the respective notches 222. Additionally, as shown, the protrusion 212 includes a coating surface 260 that is generally perpendicular to the hole base 226.

[0136] also, Fig.11A and Fig. 11B Further aspects of the base and cover for use in an assembly according to the present disclosure are shown. Fig.11A As shown, the microtiter assembly 100 is shown to have a base 102 having a plurality of holes 104. In addition, Fig.11A A cover 110 is shown having a plurality of protrusions (not shown) as described above with respect to Figure 1A The cover 110 extends longitudinally. Fig. 11B 167. In particular, the cover 110 can include a plurality of spacers 160 disposed on an outer surface 167 thereof. The spacers 160 can be positioned around an outer edge 166 of the cover 110, and / or at a center 168 of the cover 110, or at any suitable location. The inventors have discovered that the use of such spacers 160 can prevent warping of the cover 110 by increasing downward pressure on the base 102 via the cover 110 to provide more accurate test results and achieve sufficient sample volume consistency on the base 102, thereby resulting in a more consistent oxygen consumption rate across the base 102.

[0137] like Fig. 11B As shown, a plurality of spacers 160 may be present in a first outer region 161 of the cover 110, a plurality of spacers 160 may be present in a second outer region 162 of the cover 110, and a single spacer 160 may be located in a central region 163 of the cover 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 warpage of the cover 110. Furthermore, the spacers 160 may be injection molded during formation of the cover 110 and may be formed of the same material as the remainder of the cover 110.

[0138] Furthermore, it should be understood that the spacers 160 can have varying thicknesses in the y-direction in order to provide uniformity and reduced warping of the cover 110. However, in other embodiments, the spacers 160 can each have the same thickness. For example, in some embodiments, the thickness of each of the spacers 160 can range from about 100 microns to about 500 microns, such as about 125 microns to about 475 microns, such as about 150 microns to about 450 microns.

[0139] In a particular embodiment, the thickness of the spacer 160 in the central region 163 can be greater than the thickness of the spacer 160 in the first outer region 161 and the second outer region 162 to provide increased downward pressure at the center of the cover 110 where more warping may occur. In such an embodiment, the thickness of the spacer 160 in the central region can range from about 175 microns to about 500 microns, such as about 200 microns to about 475 microns, such as about 225 microns to about 450 microns, while the thickness of the spacer 160 in the first outer region 161 and the second outer region 162 can range from about 100 microns to about 250 microns, such as about 125 microns to about 225 microns, such as about 150 microns to about 200 microns.

[0140] like Fig.12 As shown, Fig.11A and Fig. 11B A comparison of the coefficient of variation of oxygen consumption measurements of the microtiter plate assembly 100 using wells of the cover 110 with spacers 160 of different thicknesses compared to the cover 110 without a spacer shows that the coefficient of variation (%CV) is reduced when the spacer 160 is used compared to when the spacer is not used.

[0141] Aspects of the present disclosure can be further understood in light of the following non-limiting examples. Example 1

[0142] A cover according to the present disclosure was formed and positioned into the corresponding wells by allowing the protrusions to sink into the corresponding wells and then pushing the cover into place, thereby allowing controlled bubble dissipation. The sample was perfused with nitrogen using Mocon's or OpTech or oxygen sensitive phosphorescent probes ( Oxygen ingress was tested by monitoring the deoxygenation level using a CLARIOstar Xtra to show reoxygenation of fully deoxygenated samples; and oxygen ingress was measured using a fluorescence plate reader (CLARIOstar BMG Labtech, where the plate reader software reports the probe emission intensity or lifetime as defined above). To maintain sample deoxygenation, plate preparation was performed in a hypoxic bag flushed and filled with nitrogen. Negative (100% air saturation) and positive (0% O2) controls were added as well as samples containing an oil layer in the headspace.

[0143] like Figure 5A As shown, two samples with a cap-seal / cap according to the present disclosure were tested, one of which had radial recesses filled with paraffin. Figure 5A As shown, the caps with wax exhibited similar reoxygenation to the caps without wax, indicating that the 1.75 mm 2 The radial notch of the radial cross section and the distal end inclined at an angle of 5° do not play a significant role in oxygen back diffusion. Similarly, Figure 5B Contains according to Figure 5A Two samples were prepared, the difference being that the well and base included an additional glass coating. Figure 5B It was shown that even when the microplate wells were coated with a glass coating to reduce reoxygenation from the lid and well base, the radial notches did not significantly contribute to oxygen back diffusion.

[0144] also, Figure 5C A comparison of oxygen ingress from coated and uncoated microplate wells is shown. Figure 5C As shown, the cap formed according to the present disclosure exhibits a low oxygen back diffusion rate. Figure 5A-Figure 5C As shown, samples formed according to the present disclosure effectively dissipated gas bubbles and had back diffusion comparable to the oil headspace samples. Example 2

[0145] Formed with 1.75mm 2 A cover according to the present disclosure having a radial notch in a radial cross section and a distal end inclined at angles of 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. Figure 6 Shown is the oxygen consumption of A549 cells seeded at a density of 65,000 cells and a filling volume of 50 μL. Figure 6 As shown, the cap formed according to the present disclosure was more sensitive to respiration, showing a 5-fold increase in the rate of change of the probe signal, and showed complete deoxygenation within the target time of 45 minutes (as shown, completed within 30 minutes, which is within the 45 minute target), compared to the oil headspace sample. Example 3

[0146] A sample according to the present disclosure was formed having the following dimensions:

[0147] like Figure 7 As shown, benchmark tests were performed against an oil headspace control and an XF flux analyzer according to embodiments of the present disclosure. Figure 7 As shown, even at very low seeding densities, the cap according to the present disclosure exhibits similar sensitivity to the Seahorse analyzer, and much higher sensitivity compared to oil headspace samples. Example 4

[0148] Interrogation was performed in the same manner as in Example 1 above, except that a solid-state sensor containing an oxygen-sensitive photoluminescent dye in an oxygen-permeable carrier matrix was used, which was deposited on the inclined distal tip of the protrusion. As described above, a plate reader (BioTek Cytation 5 from Agilent Technologies) was used to interrogate the samples. Cell samples of 40,000 A549 cells per well were used in F12K medium. Figure 8 As shown, the solid-state sensor deposited on the cover according to the present disclosure exhibited good oxygen consumption sensing over a 1 hour measurement with an average rate of 5.89 μs / h and a %CV of 12.82. Example 5

[0149] Interrogation was performed in the same manner as in Example 1 above, except that changes in both pH and oxygen consumption were measured. Measurements were performed on a BioTek Cytation 5 and BMG Clariostar plate reader as discussed above. Fig. 9 A pH calibration was obtained as shown, demonstrating compatibility with a cap according to the present disclosure.

[0150] A cell sample of 30,000 A549 cells, rotenone, antimycin A and XF DMEM medium with 5 mM HEPES were used in combination with the cap and method according to Example 1. Both pH and oxygen consumption were measured simultaneously in the sample. Fig. 10A (pH) and Fig. 10B (oxygen consumption), the caps of the present disclosure exhibit sensitive measurement of oxygen consumption during multiplexing, as shown in this example at pH. Example 6

[0151] Cell samples of 30,000 A549 cells were plated overnight and then treated with tamoxifen for 1 hour or 24 hours in a plate assembly of the present disclosure, and then the cells were assayed to determine oxygen consumption levels and cell viability. Prior to assay, the cells were loaded with calcein AM and hoescht dyes. The cell culture medium was replaced with a culture medium containing MitoXpress Xtra, and then the cells were imaged using a GFP LED / filter kit using a Cytation 5. The MitoXpress Xtra assay was initiated by inserting the cover of the present disclosure onto the base of the plate assembly.

[0152] like Figure 13-14As shown, the ability to multiplex cell viability and mitochondrial respiration measurements is demonstrated and context is added to the data. It allows the user to determine whether a compound or drug directly affects mitochondria (because mitochondrial inhibitors will be revealed by a sharp decrease in oxygen consumption), or whether this effect is due to widespread cytotoxicity. The loss of viability is revealed by calcein AM. When cells are treated for 24h, the assay can also be used to determine the overall metabolic flexibility of the cells. If respiration is reduced but viability is maintained, the cells can adapt to produce ATP via glycolysis in addition to OXPHOS. Example 7

[0153] Cells were loaded with MitoTracker and Hoechst according to the manufacturer's instructions. Cells were washed with culture medium and then the culture medium was replaced with culture medium containing MitoXpress Xtra. Using Cytation 5 and RFP LED / filter kit, imaging was performed while the plate was incubated with the compound. The cover of the present disclosure was then placed on the base of the plate assembly and the MitoXpress assay was run as usual.

[0154] Fig.15 The effects of the compounds on the mitochondrial network are shown. Rotenone, antimycin and oligomycin cause the network to condense and fragment compared to the control. The effects on the structure are then reflected in the oxygen consumption rate, showing a direct correlation between structure and function. Different compounds lead to different effects. Some promote fusion, some inhibit fusion, some promote fission, and some restrict fission. The new insight from this approach is that for the first time changes in mitochondrial structure can be linked to functional changes in respiration, such as Fig.16 shown in the same well. Example 8

[0155] Cells were loaded with 2.5 μM JC-1 and Hoechst for 45 min. Cells were washed 3x in culture medium and initial images were acquired using a Cytation 5 using the GFP and RFP LED / filter sets. Culture medium was replaced with that containing MitoXpress Xtra. Cells were treated with compounds for 1 h and then imaged as Fig.17 As shown. Respirometry was started by inserting the cover of the present disclosure onto the base of the plate assembly. Oxygen consumption of cells was measured using a MitoXpress Xtra filter cube and 2 monochromator measurements (red 475ex, 535em and green 475ex 590em). JC-1 ratio was calculated by red FI / green FI.

[0156] Simultaneous measurements of mitochondrial membrane potential (MMP) and oxygen consumption from the same well are shown. Fig.17 and Fig.18It was shown that the conventional method of measuring JC-1 using open well measurements was comparable to that with the lid in the well, but was improved in the case of the lid because the background signal was reduced. Fig.18 The fluorescent images in the figure show JC-1 plus Hoechst staining in living cells before and after treatment. Fig.19 Oxygen consumption traces and oxygen consumption rate data from compound-treated cells are shown. Fig. 20 Kinetic JC-1 data reported on MMP using PMT measurements of JC-1 ratios from the same wells as the MitoXpress Xtra data are shown. The control reacted as expected. The assay is able to detect changes in hyperpolarization, depolarization, and basal levels, as well as to correlate changes in MMP with changes in mitochondrial respiration rate. The method can distinguish between membrane potential loss due to uncoupling and due to inhibition of the electron transport chain, which would otherwise be undetectable. Example 9

[0157] The cells are counted and resuspended in culture medium with MitoXpress Xtra reagent. They are then plated on PDL-coated microplates followed by a centrifugation step. The cells are treated with the compound, the lid of the disclosure is then inserted, and the measurement in the plate reader is started. Fig.21 showed that THP-1 cells grown in suspension responded to the control compound as expected. This validates the method used to attach cells to the PDL plates and Xtra is suitable for measuring respiration. Example 10

[0158] Cells were loaded with 2.5 μM JC-1 and Hoechst for 45 min. Cells were washed 3x in culture medium and initial images were acquired using a Cytation 5 using the GFP and RFP LED / filter sets. Red FI / Green FI Fig.23 As with the adherent cell assay, suspension cells can also be loaded with JC-1 and imaged. Cells respond to control compounds as expected. Cells can be imaged after the MitoXpress Xtra assay without disturbing the monolayer, as shown in Figure 22A-22D shown.

[0159] These and other modifications and variations of the present disclosure may be implemented by those of ordinary skill in the art without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. In addition, it should be understood that aspects of the various embodiments may be interchangeable in whole or in part. In addition, it will be appreciated by those of ordinary skill in the art that the foregoing description is by way of example only and is not intended to limit the disclosure so further described in the appended claims.

Claims

1. A microtiter plate assembly, comprising: a base including at least one hole; as well as a cover including at least one translucent protrusion corresponding to the at least one hole; wherein the at least one translucent protrusion extends from a proximal end adjacent the cover to a distal end having a radial notch and an angled tip having an angle of about 5° or more relative to a plane of the cover; and wherein a wall gap is defined between a side wall of the at least one protrusion and a side wall of the at least one corresponding hole, and wherein a radial notch gap is defined between an apex of the radial notch and a side wall of the corresponding hole, wherein the wall gap is 50% or less of the radial notch gap.

2. A microtiter plate assembly, comprising: a base comprising a plurality of apertures spaced apart in an array, wherein each aperture is adjacent to a second aperture; as well as a cover comprising a plurality of translucent protrusions spaced apart in an array corresponding to the array of the plurality of apertures, wherein each protrusion is adjacent to a second protrusion; wherein the plurality of translucent protrusions extend from a proximal end adjacent the cover to a distal end having a radial notch and an angled tip, the angled tip having an angle of about 5° or more relative to a plane of the cover; and wherein a height gap is defined between a distal-most point of the distal end of each protrusion and a hole base of the corresponding hole, and wherein a percentage variation in height gap between the protrusion and the hole and an adjacent second protrusion and second hole is about 10% or less.

3. The microtiter plate assembly of claim 1 or 2, wherein at least a portion of the assembly comprises an oxygen permeability of about 600 cm 3 / m 2 / 24 hours or less, and / or wherein at least a portion of the assembly is formed of a substrate material having an oxygen permeability of about 120 cm 3 / m 2 / 24 hours or less for polymeric or ceramic composite coatings.

4. The microtiter plate assembly according to any of the preceding claims, wherein at least a portion of the assembly is coated with: Al2O3, SiO2, silicon oxide, silicon nitride, silicon oxynitride, aluminum oxide, Al2O3 alternating with polyacrylate, or a combination thereof.

5. A microtiter plate assembly according to any one of the preceding claims, wherein the radial recess has a diameter of about 0.7 mm. 2 or greater radial cross-section, preferably wherein the radial recess has a radial cross-section of about 1.25 mm 2 or larger radial cross-section.

6. The 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. The microtiter plate assembly according to any one of the preceding claims, wherein the cover is reversibly associated with the base.

8. A microtiter plate assembly according to any of the preceding claims, wherein 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, the length of the radius of the radial recess is about 5% to about 30% of the length of the radius of the cross-section of the protrusion including the radial recess.

9. 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, wherein the distal radius is about 2% to about 15% smaller than the proximal radius.

10. The microtiter plate assembly of any one of the preceding claims, wherein the assembly is configured to measure oxygen consumption in 45 minutes or less for a sample having a seeding density of 250,000 cells or less.

11. The microtiter plate assembly according to any one of the preceding claims, wherein the cover is milled, extruded, injection molded or a combination thereof.

12. 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 protrusions.

13. The microtiter plate assembly according to any one of the preceding claims, wherein the cover is a single piece.

14. The microtiter plate assembly according to any one of claims 1 to 12, wherein the cover is formed by a frame and one or more rows comprising a plurality of translucent protrusions attached to the frame.

15. The microtiter plate assembly of claim 14, wherein the cover comprises eight rows, each row comprising twelve corresponding protrusions.

16. A microtiter plate assembly according to any one of the preceding claims, wherein at least a portion of the assembly is formed from an acrylate polymer.

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

18. A method of measuring oxygen consumption rate using an assembly according to any one of the preceding claims, the method comprising: placing a sample and an oxygen sensitive probe into the at least one well, contacting the sample with the corresponding protrusion, and The oxygen consumption was measured using a fluorescence plate reader.

19. The method of claim 18, further comprising measuring acidification.

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

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

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