Patent Description:
In the field of cell analysis, cells are commonly placed in a multiwell microplate for purposes of testing multiple conditions and replicates in a single experiment. Standard microplates, such as <NUM>- and <NUM>-well plates, are two-dimensional arrays of wells. Such arrays include some wells that are at the border or edge of the array, i.e., in the first row, first column, last row, or last column. Border wells and non-border wells can experience different conditions; this is commonly known as an "edge effect". Because such assays are typically conducted at mammalian body temperature (<NUM>), and border wells are more exposed to the external environment, the environment within the border wells may be substantially different from that of the non-border wells. The evaporation of liquid from wells adjacent to the border of the plate occurs at a higher rate than that of non-border wells. This causes a temperature drop in the border wells due to evaporative cooling, resulting in an increase in the concentration of solutes in the liquid. Both the temperature differences and the concentration difference contribute to data inconsistency in these types of assays. Live-cell assays are particularly sensitive to these effects due to the dynamic nature of the assay and the sensitivity of living, metabolically active cells to the environmental conditions in which they are being measured. Examples of these types of assays include FLIPR calcium flux assays, Corning EPIC label-free assays, and certain high-content imaging assays.

Several solutions have been proposed and applied to such standard microplates to address this problem. One workaround is to sacrifice the use of the border wells in the assay. By simply filling them with fluid to the same height as the assay wells, the border wells provide a humidity buffer. This approach has serious drawbacks in that the capacity of the microplate is significantly diminished, and in the case of a <NUM>-well plate more than half of the wells are sacrificed. As the size of the well array in the microplate decreases, a higher fraction of wells become border wells. At the extreme, in one-dimensional arrays, every well has a high rate of evaporation.

Another workaround is to seal the wells or plate by overlaying the assay wells with oil or wrapping the covered plate with a plastic paraffin film, such as Parafilm M® film available from Bemis Company, Inc. , or similar material. One of the drawbacks to these methods is that gas exchange is reduced. Metabolically active cells require oxygen; thus restricting the supply of oxygen can be detrimental to the cells and cause changes in assay results.

Existing solutions to this problem include modifications to the instrumentation or the cell growth vessel, i.e., microplate and cover. A few instrumentation manufacturers attempt to mitigate these effects by putting humidity control into the measuring chambers in which the microplate is placed. In general, however, these options are rare as high humidity levels can cause problems with the instrument electronics.

Modifications to the cell growth vessel may include changes to the design of the microplate and lid. Changes to the lid include adding a moisture-holding layer to the lid. However, in the case of live-cell assays where addition of reagent during the course of the assay is required, a lid or cover cannot be used.

The addition of perimeter or border wells to the microplate provides an environmental buffer between the assay well and the ambient laboratory conditions. For example, a plate may have large edge troughs, e.g., four troughs, surrounding the array of wells. Fluid may be placed in each trough, thus providing an environmental buffer. A potential drawback of this design is the large volume of each trough. Because well plates are shallow, there is potential sloshing of the border fluid when the plate is tilted or moved around the laboratory. In addition, the depth of the troughs, being the same depth as that of the wells, may require that a significant amount of fluid, more than 10x the volume of the assay well, be added to each trough. Therefore the operator may need to use a different tool (such as a different volume pipet) to fill the border troughs and the assay wells.

Standard microplate designs include a lid or cover where the edge or skirt of the cover can be up to half the height of the plate itself and protrudes <NUM>-<NUM> millimeters ("mm") beyond the wall of the plate. This may present a problem while handling these plates, as it takes some dexterity to consistently pick up both the plate and the lid off of a surface, e.g., to avoid accidentally picking up only the lid and thus exposing the contents of the plate. When dealing with cell cultures that must be maintained under sterile conditions, current plate and cover assembly designs introduce considerable risk to the integrity of the cultures. Similar risks apply to assays where the contents of the wells must be protected from ambient light.

Standard microplate designs have a fixed height and footprint, such that the volume of the wells varies with the number of wells arrayed in the plate. For example, a standard <NUM>-well plate has four times as many wells as a standard <NUM>-well plate, but each well is approximately one-fourth the volume. Likewise, as well density (i.e., wells per plate) goes down, the volume per well increases. This design, although convenient for maintaining a standard footprint, requires that the researcher use more cells and reagents per well when using a lower-density plate. In addition, the spacing between wells changes, which can be an inconvenience when adding reagents to the assay plate.

Presently, no microplate is commercially available for performing an assay on a fewer number of wells while maintaining standard volumes and well-to-well spacing. Maintaining these features and reducing the number of wells may require reducing the footprint. However, since many standard laboratory workflows and instruments are designed to this standard, an adapter or carrier of some sort would be required. Examples of instruments that accept standard-footprint microplates include plate readers, high content imaging systems, centrifuges, and automated plate handling robots.

Microscope slides adhere to a different standard in the lab, and some products exist that bridge the microplate and slide formats. Some commercially available slides contain assay wells fused to a glass microscope slide, providing assay wells with glass bottoms designed for high-resolution imaging on microscopes. Although they do provide wells, the dimensions of the wells vary and are not standard with respect to well-to-well spacing nor length and width dimensions.

A commercially available carrier for microscope slides that conforms to the Society for Laboratory Automation and Screening ("SLAS") microplate footprint and height standards is designed for imaging applications, but the placement of the slides in the carrier allows for some variability in well position, which may make automated analysis challenging.

<CIT> relates to a container with a base body, comprising a base plate and side walls standing out therefrom in an at least approximately perpendicular arrangement, and with wells disposed in the base body. The wells are provided in the form of a recess in the base plate and the side walls of the base plate are disposed in at least the approximately opposite direction from the recesses in order to accommodate a volume.

<CIT> discloses a system including a support that holds multiple sample receptacles, computational circuitry to execute a protocol script that delivers instructions for executing various steps of the procedure, and a progress sensor that advances the execution of the protocol script. Also disclosed is a system for storing and assembling a plurality or reagents comprising a frame having a plurality of differently sized or shaped cavities, the frame having a microplate footprint; and a plurality of inserts sized to be inserted into the plurality of cavities, at least one insert comprising at least one fluid receiving member.

<CIT> discloses devices and methods that measure one or more properties of a living cell culture that is contained in liquid media within a vessel, and typically analyzes plural cell cultures contained in plural vessels such as the wells of a multiwell microplate substantially in parallel. The devices incorporate a sensor that remains in equilibrium with, e.g., remains submerged within, the liquid cell media during the performance of a measurement and during addition of one or more cell affecting fluids such as solutions of potential drug compounds. One embodiment comprises a cartridge and microplate in the form of a typical multiwell microplate. The cartridge comprises a frame with a planar surface defining a plurality of reagents that correspond to i.e. register with a number of the respective openings of the plurality of wells defined in the multiwell plate. Within each of these regions, four ports which serve as test compound reservoirs and a central aperture to a sleeve having one or more sensors are defined.

Disclosed is a multiwell microplate for holding liquid samples. The multiwell microplate includes a frame defining a plurality of wells disposed in a single column, each well having an opening with a length l<NUM>; a moat disposed about the plurality of wells; and a plurality of walls traversing the moat. The walls define a plurality of compartments, each compartment having a length l<NUM> selected from a range of greater than l<NUM> and less than <NUM><NUM>.

One or more of the following features may be included. The well length l<NUM> may be selected from a range of <NUM> to <NUM> (<NUM> to <NUM> in). The plurality of wells may include eight wells. The moat may include eight compartments.

Two compartments disposed on opposing sides of the single column of wells may be in fluidic communication via an equalizer channel. A depth of the two compartments in communication via the equalizer channel may be less than a depth of compartments adjacent thereto.

A depth of at least one compartment may be less than a depth of one of the wells, e.g., the depth of the at least one compartment may be up to <NUM>% of the depth of one of the wells. A depth of a compartment proximate an end portion of the frame may be less than a depth of a compartment disposed at a center portion of the frame. All of the compartments may have a substantially equal length.

A lifting tab may be defined on an end portion of the frame. At least one well may be opaque white or opaque black. The frame may define an indent on a lower edge.

The invention comprises a multiwell microplate carrier, as defined in claim <NUM>, including a body defining a plurality of regions configured to hold a plurality of multiwell microplates in parallel, each multiwell microplate defining a single column of wells, and each of the regions defining a plurality of openings adapted to mate with the single columns of wells, as well as a collar for surrounding a bottom region of each microplate well when installed, wherein each collar forms an opening for positioning and light blockage.

One or more of the following features may be included. The body may have a base footprint with outside dimensions of approximately <NUM> inches (<NUM>) by <NUM> inches (<NUM>). Each region may define eight openings. The body may define three or four regions configured to hold three or four multiwell microplates, respectively.

Further disclosed is a cartridge for mating with the multiwell microplate described herein. The cartridge includes a substantially planar surface having a plurality of regions corresponding to a number of respective openings of the wells in the multiwall microplate. Also located in plural respective regions of the cartridge is a sensor or a portion of a sensor adapted to analyze a constituent in a well and/or an aperture adapted to receive a sensor. At least one port may be formed in the cartridge, the port being adapted to deliver a test fluid to a respective well of the plate. The multiwell microplate may include eight wells and the cartridge may include eight regions.

Further disclosed, but not forming part of the invention, is a method for preparing a liquid analytical sample. The method includes delivering the analytical sample to a well defined by a frame of a multiwell microplate. A fluid is delivered to a moat defined by the frame. The frame defines a plurality of wells disposed in a single column, each well having an opening with a length l<NUM>. The moat is disposed about the plurality of wells. A plurality of walls traverses the moat, the walls defining a plurality of compartments, each compartment having a length l<NUM> selected from a range of greater than l<NUM> and less than <NUM><NUM>.

One or more of the following features may be included. Delivering the analytical sample to the well may include using a pipettor. Delivering the fluid to the moat may include using a pipettor.

The invention further relates to a kit comprising the multiwell microplate carrier of the invention as well as the multiwell microplate disclosed herein. The kit may further comprise the cartridge disclosed herewith.

Evaporation from peripheral wells of a multiwell microplate may have a negative impact on various analytical steps, including cell seeding, cell plate incubation and running assays. In particular, cell-based assays ("CBA") with adherent cells are susceptible to edge effects from cell seeding and cell plate incubation. Live-cells assays such as label-free and extracellular flux ("XF") measurements are also susceptible to edge effects during the running of the assays. Multiwell plate designs having moats with compartments to hold hydration fluid, e.g., water or cell media, at and/or near the edges of the multiwell plate, help reduce such edge effects, reducing the evaporation of fluid from the wells by providing a humidified buffer between the air above the wells and the drier air outside a perimeter of the plate.

Referring to <FIG> and <FIG>, a multiwell microplate <NUM> is formed from a frame <NUM> defining a single column of wells <NUM>. The number of wells <NUM> in a plate may vary from two to thousands, preferably a maximum of <NUM> (corresponding to an industry standard of wellplates with <NUM> wells, with <NUM> wells in a single column). The multiwell microplate may have a column of four, six, or twelve wells. The multiwell microplate has eight wells <NUM>. A configuration with eight wells may be especially advantageous, as it allows up to four replicates of two conditions such as disease/normal, drug treated/native, or genetic knock-out vs. wild type, while maintaining a small footprint. Moreover, many analytical instruments are configured to handle well plates having columns of eight wells, such as <NUM> well plates (<NUM> x <NUM>).

The multiwell microplate <NUM> includes a one-dimensional pattern of wells complying, in relevant part, with the pattern and dimensions of a microplate, as described by the American National Standards Institute and Society for Laboratory Automation and Screening standards, including Height Dimensions for Microplates (ANSI/SLAS <NUM>-<NUM>, <NUM>/<NUM>/<NUM>); Well Positions for Microplates (ANSI/SLAS <NUM>/<NUM>, <NUM>/<NUM>/<NUM>); and Footprint Dimensions for Microplates (ANSI/SLAS <NUM>-<NUM>, <NUM>/<NUM>/<NUM>).

The multiwell microplate may be formed from a molded plastic, such as polystyrene, polypropylene, polycarbonate, or other suitable material. The bottoms of the wells may be transparent and the sides colored black to reduce optical cross-talk from one well to another. , for use with luminescence measurements, the wells may be white. , for use in high-resolution imaging applications, the plate may be formed with glass as the bottom of the wells and plastic polymer forming the sides of the plate and walls of the wells.

Each of the wells may have a top portion with an opening having a length l<NUM> as well as a bottom portion that may be cylindrical or square, and may have a tapered sidewall. A seating surface may be provided to act as a positive stop for sensors disposed on barriers (see discussion of cartridge with respect to <FIG> and <FIG>). This seating surface enables the creation of a localized reduced volume of medium, as discussed in <CIT> The seating surface may be defined by a plurality of raised dots, e.g., three dots, on a bottom surface of a well. The well length l<NUM> can be any dimension and may be preferably selected from a range of <NUM> to <NUM>, e.g., <NUM>. Preferably, the wells are spaced equally from each other, e.g., <NUM> - <NUM>, more preferably <NUM> as measured center to center of the wells. Each of the wells in the microwell plate can have substantially the same dimensions, including the same well length l<NUM> as well as a width equal to the length. However, the wells may have varying dimensions, including different well lengths l<NUM>. A depth of the wells may range from <NUM> to <NUM> or more, preferably about <NUM>.

A moat <NUM> efxtends about an external perimeter of the wells. A plurality of walls <NUM> traverse the moat, the walls <NUM> defining a plurality of compartments <NUM>. The walls <NUM> are preferably thick enough to provide rigidity to the microplate, while being thin enough to be injection molded without distortion. Accordingly, a thickness of the walls may range from <NUM> to <NUM>, preferably about <NUM>. The compartments each have a length l<NUM> that is preferably a multiple of l<NUM> and less than <NUM><NUM>, preferably about <NUM><NUM>, and not less than <NUM>. For example, if a well opening has a length l<NUM> of <NUM>, an abutting compartment may have a length of <NUM><NUM> of <NUM>. A length of less than <NUM> (<NUM> well-to-well spacing) could make filling the compartments challenging. All of the compartments may have substantially equal longitudinal lengths, i.e., the length from one end wall to an opposing end wall varying no more than <NUM>%.

The moat has eight compartments and eight wells, with one or more compartments having a length approximately equal to the sum of the lengths of approximately two well openings, plus a thickness of one or more walls defining the well openings.

Two compartments disposed on opposing sides of the single column of wells may be in fluidic communication via an equalizer channel <NUM>. The moat may include two equalizer channels <NUM>, one at each end of the multiwell microplate. To equalize the volumes of the compartments of the moat, a depth of two compartments in communication via the equalizer channel may be less than a depth of compartments adjacent thereto. The equalizer channel may be disposed at an end of the multiwell microplate, and is <NUM> inches (<NUM>) wide and <NUM> inches (<NUM>) deep. The dimensions of the equalizer channel are preferably small enough to reduce the contribution of the channel width to the overall plate size but are wide enough to overcome surface tension and allow the chosen fluid to fill the channel. The channel may have a feature <NUM> (e.g., surface tension breaker <NUM> as illustrated in <FIG>) that breaks the surface tension of the fluid allowing it to self-fill at a lower volume. Since sharp corners break the surface tension of the fluid, to stimlate fluid flow through the narrow opening of the equalizer channel, one or more sharp edges may be included.

A depth of at least one compartment may be less than a depth of one of the wells, e.g., the depth of the at least one compartment may be <NUM>% or less than the depth of one of the wells.

A depth of a compartment proximate an end portion of the frame may be less than a depth of a compartment disposed closer to a center portion of the frame. To maintain a constant fluid height across all compartments with <NUM>µl in end compartments connected by an equalizer channel and <NUM>µl of fluid in the inner compartments, the inner compartments may be <NUM> inches (<NUM>,<NUM>) deeper than the outer compartments.

The moat may have a width of at least <NUM> inches (<NUM>) and no more than <NUM> inches (<NUM>), preferably approximately <NUM> inches (<NUM>). A moat that is too narrow could minimize the benefit of having a hydrating barrier between the wells and the dry outside air; whereas, a moat that is too wide could introduce the risk of sloshing and contamination of the assay wells.

All of the compartments may be of substantially equal length, e.g., varying no more than <NUM>%.

Various features of the moat facilitate its filling with a multi-channel pipettor design for Society for Biomolecular Screening ("SBS") standard microplates. Suitable multi-channel pipettors include Eppendorf <NUM> and Mettler-Toledo L8-200XLS+, available from Eppendorf AG and Mettler-Toledor International Inc. , respectively. The walls defining compartments are positioned so as to not interfere with pipette tips on the multi-channel pipettor. Such multi-channel pipettors have a standard tip-to-tip spacing of <NUM>, so compartments of a moat preferably allow access of an equal number of pipet tips into each compartment. Equalizer channels at the ends allow fluid to be drawn from the side compartments, thereby enabling hydration fluids to surround the end wells. The compartments are preferably more than one well and less than six wells in length to reduce splashing of liquid out of the microwell plate or contamination of assay wells with hydration liquid. Finally, the moat depth is preferably <NUM>% or less than the well depth to reduce the required volume of hydration liquid and to allow the use of a pipettor the same size as a cell pipettor.

A lifting tab <NUM> may be defined on one or both end portions of the frame. The lifting tab may have a length l<NUM> of <NUM> to <NUM> inches (<NUM> to <NUM>), e.g., <NUM> inches (<NUM>). The lifting tab facilitates lifting of the multiwell microplate and a cover or a microplate and a cartridge, without removing the cover or cartridge.

The lower edge of the frame may define one or more indents <NUM>. The indents may be positioned at the ends and/or the sides of the frame. The incorporation of one or more indents provides stability for the frame when positioned in a carrier tray. Moreover, without the indents, the frame would sit higher in the carrier, which may prevent its use in different instrumentation. The height of one multiwell microplate is preferably about <NUM> to <NUM> inches (<NUM> to <NUM>), more preferably <NUM> inches (<NUM>) without the carrier. Side-loading plate readers, for example, have plate access heights of <NUM> to <NUM>. The indent allows placement of the plates in the carrier with minimal added height (<NUM> to <NUM> inches, i.e., <NUM> to <NUM>). The carrier adds less than <NUM> inches (<NUM>) to the height of the plate.

The relative surface areas of fluids in the compartments and the wells are relevant for the impact of the moat on reducing evaporation in the wells. If the surface area of the fluid in the compartments is too small, the reduction of evaporation in the wells may be negligible. If the surface area of the fluid in the compartments is larger than necessary for the desired impact, the multiwell microplate may be less compact than necessary, and may present a challenge in filling the compartments with the same pipettes that are used for filling the wells.

Examples disclosed may provide the surface areas and volumes when fluid is introduced into the wells and compartments indicated in Table <NUM>. The disclosure includes ranges of the preferred values of at least ±<NUM>% and greater; preferably the ratios of volumes and surface areas of the wells and compartments are substantially equal to the indicated values, i.e., ±<NUM>%. The difference between the two bottom-up measurements in the compartments for the cell culture and assay conditions may be <NUM> inches (<NUM>). This difference in depth results in the fluid height of all compartments being at a constant depth relative to the top surface of the plate (i.e., <NUM> inches <NUM>). This difference compensates for the equalizer channel.

Referring to <FIG> and <FIG>, a cartridge <NUM> is configured to mate with the multiwell microplate <NUM>. The cartridge <NUM> has a generally planar surface <NUM> including a cartridge frame made, e.g., from molded plastic, such as polystyrene, polypropylene, polycarbonate, or other suitable material. Planar surface <NUM> defines a plurality of regions <NUM> that correspond to, i.e., register or mate with, a number of the respective openings of a plurality of wells <NUM> defined in the multiwell microplate <NUM>. Within each of these regions <NUM>, the planar surface defines first, second, third, and fourth ports <NUM>, which serve as test compound reservoirs, and a central aperture <NUM> to a sleeve <NUM>. Each of the ports is adapted to hold and to release on demand a test fluid to the respective well <NUM> beneath it. The ports <NUM> are sized and positioned so that groups of four ports may be positioned over each well <NUM> and test fluid from any one of the four ports may be delivered to a respective well <NUM>. The number of ports in each region may be less than four or greater than four. The ports <NUM> and sleeves <NUM> may be compliantly mounted relative to the multiwell microplate <NUM> so as to permit them to nest within the microplate by accommodating lateral movement. The construction of the cartridge to include compliant regions permits its manufacture to looser tolerances, and permits the cartridge to be used with slightly differently dimensioned microplates. Compliance can be achieved, for example, by using an elastomeric polymer to form planar element <NUM>, so as to permit relative movement between the frame <NUM> and the sleeves and ports in each region.

Each of the ports <NUM> may have a cylindrical, conic or cubic shape, open at planar surface <NUM> at the top and closed at the bottom except for a small hole, i.e., a capillary aperture, typically centered within the bottom surface. The capillary aperture is adapted to retain test fluid in the port, e.g., by surface tension, absent an external force, such as a positive pressure differential force, a negative pressure differential force, or alternatively a centrifugal force. Each port may be fabricated from a polymer material that is impervious to test compounds, or from any other suitable solid material, e.g., aluminum. When configured for use with a multiwell microplate <NUM>, the liquid volume contained by each port may range from <NUM>µl to as little as <NUM>µl, although volumes outside this range can be utilized.

Referring to <FIG>, in each region of the cartridge <NUM>, disposed between and associated with one or more ports <NUM>, is the submersible sensor sleeve <NUM> or barrier, adapted to be disposed in the corresponding well <NUM>. Sensor sleeve <NUM> may have one or more sensors <NUM> disposed on a lower surface <NUM> thereof for insertion into media in a well <NUM>. One example of a sensor for this purpose is a fluorescent indicator, such as an oxygen-quenched fluorophore, embedded in an oxygen permeable substance, such as silicone rubber. The fluorophore has fluorescent properties dependent on the presence and/or concentration of a constituent in the well <NUM>. Other types of known sensors may be used, such as electrochemical sensors, Clark electrodes, etc. Sensor sleeve <NUM> may define an aperture and an internal volume adapted to receive a sensor.

The cartridge <NUM> may be attached to the sensor sleeve, or may be located proximal to the sleeve without attachment, to allow independent movement. The cartridge <NUM> may include an array of compound storage and delivery ports assembled into a single unit and associated with a similar array of sensor sleeves.

Referring to <FIG>, the cartridge <NUM> is sized and shaped to mate with multiwell microplate <NUM>. Accordingly, if the microplate has eight wells, the cartridge has eight sleeves.

Referring to <FIG>, the apparatus may also feature a removable cover <NUM> for the cartridge <NUM> and/or for the multiwell microplate <NUM>. The cover <NUM> may be configured to fit over the cartridge <NUM>, thereby to reduce possible contamination or evaporation of fluids disposed in the ports <NUM> of the cartridge. The cover <NUM> may also be configured to fit directly over the multiwell microplate <NUM>, to help protect the contents of the wells and compartments when the microplate <NUM> is not in contact or mated with the cartridge <NUM>.

Referring to <FIG> and <FIG>, a multiwell microplate carrier tray <NUM> allows several, e.g., three or four, single-column multiwell microplates to be placed and measured in an instrument designed for <NUM> well standard microplates that comply with standard ANSI/SLAS <NUM>-<NUM>. Accordingly, the carrier tray may have outer dimensions of <NUM> inches (<NUM>) ± <NUM> inches (<NUM>) by <NUM> inches (<NUM>) ± <NUM> inches (<NUM>), i.e., about <NUM> by <NUM> inches or about <NUM> x <NUM>. In other embodiments, the outer dimensions of the carrier tray may be scaled, depending upon the number of wells in the single-column microplates and the instrument in which measurements may be carried out.

In one preferred embodiment, the carrier has three regions <NUM> defining a plurality of openings <NUM> configured to align and mate with the wells of each multiwell microplate <NUM>. In one preferred embodiment, in use, the columns of wells of the multiwell microplates are disposed at positions that correspond to columns <NUM>, <NUM>, and <NUM> of a <NUM>-well microplate. Since the wells of the disclosed multiwell microplates are located at positions defined by the ANSI/SLAS standard, no modification of the plate readers is required. A collar <NUM> surrounds the bottom region of each microplate well when installed in the cartridge. Each collar forms a circular opening that provides positioning as well as light blockage. The collar may be colored black to shield crosstalk light from fluorescent signaling molecules in wells, or may be white to amplify emitted light from luminescent markers. The carrier may include slots <NUM> that correspond to indents on the multiwell microplate. The skirts of two adjacent microplates may fit into each slot. Scalloped edges <NUM> enable a user to easily remove the microplates as necessary, while providing rigidity to the carrier.

In one preferred embodiment, the carrier openings allow the microplate to sit in the carrier at the same height as if the plate was not in the carrier, i.e., the height of the plate is equal to the height of the plate and carrier assembly.

Cartridges <NUM> and covers <NUM> may be placed over the microplates <NUM>, as discussed above. The multiwell microplates and cartridges may generally be used as described in <CIT> and <CIT>. Moreover, the individual wells, barriers, and ports may have any of the characteristics and features of the wells, barriers, and ports described in these patents.

In use, a liquid analytical sample may be prepared by delivering the analytical sample to a well defined by a frame of a multiwell microplate <NUM>, and delivering a fluid to a moat <NUM> defined by the frame. The analytical sample may be, for example, cells in a media. The fluid may be the same media, or another liquid, such as water. Both the analytical sample and the fluid may be delivered by a pipettor; in some embodiments, the sample and the fluid may be delivered by the same pipettor.

Incubator evaporation experiments were run to compare evaporation in covered multiwell microplates with hydration fluid in moats and without such fluid. For each of six plates, <NUM> microliters of liquid was placed in each well, and for three of those plates, <NUM> microliters of liquid was placed in each compartment of the moat. Three multiwell microplates with covers but with no liquid in moats ("dry") and three multiwell microplates with covers and with liquid in moats were incubated overnight in a humidified incubator at <NUM> in a <NUM>%CO<NUM> atmosphere. The volume of liquid remaining in each well was measured, and the following values determined:.

Evaporation of liquid from wells in uncovered microwell plates was measured after conducting a mock assay (~<NUM> minutes) within an extracellular flux analyzer instrument. Referring to <FIG>, the average % of fluid lost in a microwell plate with a filled moat was <NUM>%, whereas about <NUM>% of fluid was lost in a microwell plate with an empty moat. Evaporation is preferably reduced, as it causes variations in assay data due to changes in temperature as well as the ionic strength of the cell media.

Referring to <FIG>, cells disposed in media were observed with hydration fluid in moats and without hydration fluid. Key metabolic parameters of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were monitored in each well. The well-to-well variability in plates with dry moats (CV <NUM>-<NUM>%) was considerably higher than the variability observed for assay wells in plates with filled moats (<NUM>-<NUM>%). Low well-to-well variability of both the OCR and ECAR signals is required for good assay performance. The OCR measurement is particularly sensitive to temperature variations which can be caused by varying rates of evaporation in assay wells not protect by fluid-filled moats.

Referring to <FIG>, baseline metabolic rates (OCR and ECAR) of C2C12 cells seeded at equal densities were measured under several conditions to test the effect of the moat being filled or empty. In <FIG>, the moat was filled as prescribed (<NUM>µl per compartment) at the time of cell seeding. For plates represented by hashed bars, the moats were emptied prior to performing the assay in the XF instrument. In <FIG>, the cells were seeded and incubated overnight without placing fluid in the moats. In C and D plates represented by solid bars had fluid added to the moats prior to running the experiment. Both OCR and ECAR were measured for all plates. To assess the effect of the presence of fluid in the moats at the time of seeding on the OCR measurement, <FIG> is compared to <FIG>. Cells seeded in plates with fluid in the moats had OCR values in the range of <NUM>-<NUM>, whereas cells seeded in plates with dry moats had OCR values in the range of <NUM>-<NUM>. OCR is a measure of the metabolic health of the cells. Low OCR values indicate that the cells were not metabolically active. Similar results are seen when comparing <FIG> for the ECAR measurement. When cells are seeded in plates and the moat is not filled, the metabolic rate as measured by ECAR is also very low, indicating poor cell health. Thus, it is shown that the presence of fluid in the moats at the time of cells seeding and overnight incubation is an important requirement for good cell health in the single-column microplate.

Referring to <FIG>, inter- and intra-well variability of the background OCR signal over time was compared in a plate without fluid in the moat to a plate with fluid in the moat. For each plate tested, media was placed in each well, the plate was allowed to equilibrate in the instrument for <NUM> minutes, then measurements were made over <NUM> minutes. In the plate without fluid in the moat, the background OCR signal varied significantly from well to well, ranging from -<NUM> to +<NUM> (range of <NUM>) and rising <NUM>-<NUM> units over the <NUM> minute period. When the moat was filled, the signal was much more stable with an overall range of -<NUM> to +<NUM> (range of <NUM>) and rising about <NUM> units over the time period. Thus it is shown that the presence of fluid in the moats is required for stable background levels in this assay.

Claim 1:
A multiwell microplate carrier (<NUM>) comprising:
a body defining a plurality of regions (<NUM>) configured to hold a plurality of multiwell microplates (<NUM>) in parallel, each multiwell microplate (<NUM>) defining a single column of wells (<NUM>), and each of the regions (<NUM>) defining a plurality of openings (<NUM>) that are adapted to mate with the single columns of wells (<NUM>), characterized by
a collar (<NUM>) for surrounding a bottom region of each microplate well (<NUM>) when installed, wherein each collar (<NUM>) forms an opening for positioning and light blockage.