Cell positioning and concentrating device and process for using same

Abstract

A method and system for seeding cells on a well plate is disclosed. According to the present disclosure, a cell concentrating device is used that positions the cells at a particular location on the bottom floor of each well. The cell concentrating device improved sensitivity and uniformity when conducting analysis on the cells.

Description

CELL POSITIONING AND CONCENTRATING DEVICE AND PROCESS FOR USING SAMERelated ApplicationsThe present application claims the benefit of and priority to US Provisional Patent Application No. 63 / 663,880 having a filing date of June 25, 2025, which is incorporated herein in its entirety by reference thereto.BACKGROUND

[0001] Cellular metrology is important for research, diagnostics and therapeutics development. In some applications, stationary cells are exposed to light in order to examine and / or characterize the samples. In one aspect, for instance, a biomedical imaging and analysis instrument can be used to conduct an analysis of the type and concentration of a constituent or analyte contained in a cellular sample. These types of devices are well suited to analyzing living cells and providing useful information regarding the metabolic processes that are occurring inside the cells. For instance, the devices can provide real-time cell analyte measurements that provide a clear window into the critical functions driving cell signaling, proliferation, activation, toxicity, and / or biosynthesis.

[0002] Devices for measuring the physiological properties of biological materials, such as cells, are disclosed in U.S. Patent No. 8,697,431 and U.S. Patent No. 9,170,253, which are incorporated herein by reference. The device and method disclosed in the above patents have provided great advancements in the art. In one embodiment, for instance, fluorescence is used to interrogate a sample and to measure a constituent in the sample, such as dissolved oxygen and / or pH. During the method, a sensor material is placed in association with a light probe and contacted with a biological sample, such as living cells contained in a media. The cells typically rests on the floor of a well. The sensor material has fluorescent or chemiluminescent properties that are related to chemical or metabolic concentrations within the cell media and that are either emanating from or modified by the cells contained therein. Optical fibers are used to emit an excitation signal onto the sensor material, which then generates a fluorescent emission signal thatis sensed and analyzed for its fluorescent properties which are analyzed for determining or measuring different characteristics of the sample.

[0003] When cells are located in a well for analysis, control of the location and density of the cells is important for several reasons. Imaging and sensing rely upon cell location for consistent and reproducible measurements. In addition, the state and behavior of cells (e.g. metabolic state, respiration, etc.) can be affected by local cell density. Further, in some applications, different cell types are combined together in well-defined quantities and positions to mimic animal tissues. Positioning cells is also important for preparing cell ensembles, such as spheroid and organoid formation. Control of cell density is important to control the biology of invitro cells and cell collections.

[0004] In the past, cells were injected into a well for analysis. As the cells settled, the cells covered the entire bottom of the well. However, there are several challenges that result from having cells cover the entire bottom surface of the well, many of which are due to spatial sensitivity associated with invitro cellular assays.

[0005] Accordingly, due to the above concerns, it is often desirable for cells to be concentrated into the center of a well to avoid spatial biases, which can lead to the non-uniformity of measurements and the lack of a strong optical signal received from the cells.

[0006] In the past, attempts to control cell placement in a well involved the use of a sleeve that had an elastomeric end. The elastomeric end would be inserted into the well and form a seal with the bottom of the well. A media containing cells was then injected into the well in order to cause the cells to concentrate more towards the center of the well. Although the product has made great advances in the art, problems have been experienced in removing the sleeve once the cells have been deposited in the well. In particular, removing the sealed end from the well can cause the formation of bubbles and voids and can cause cells to become dislodged from the surface of the well.

[0007] In view of the above, a need currently exists for an improved method and system for concentrating cells at particular locations on a bottom surface of a well.SUMMARY

[0008] The present disclosure is directed to a system, device and method for positioning and concentrating cells on a surface of a well using one or more inserts that have a tubular shape and can be in the form of a funnel, such as a tapering sleeve. In accordance with the present disclosure, the inserts can be made from a single polymer material, such as through injection molding. The cell concentrating device of the present disclosure is relatively easy to insert and to remove from a well during the process of seeding cells in the well.

[0009] In one embodiment, the present disclosure is directed to a cell seeding system. The cell seeding system includes a well plate including at least one well. The well has an open end opposite a closed end and includes at least one side wall extending between the open end and the closed end. The well defines an interior volume and a depth for holding a fluid medium. The system further includes a cell concentrating device that is insertable and removable from the well. The cell concentrating device is configured to receive a cell population and direct the cell population to the closed end of the well. The cell concentrating device comprises a tubular member having a first open end defining a first opening opposite a second open end defining a second opening. The first opening is larger than the second opening. In one aspect, the tubular member can taper from the first open end to the second open end.

[0010] In one aspect, the cell concentrating device can be made from a non- elastomeric material. For instance, the cell concentrating device can be formed as a single integral structure made from a thermoplastic polymer material.

[0011] The tubular member has a shape configured to be inserted through the open end of the well. The tubular member can have a length that is at least about 90% of the depth of the well.

[0012] In one aspect, the tubular member has a length such that, when inserted into the well, does not contact a bottom surface of the well but, instead, forms a gap. The gap, for instance, can have a length of from about 5 microns to about 1 ,000 microns, such as from about 50 microns to about 500 microns, such as from about 100 microns to about 300 microns. Alternatively, the tubular member can have a length such that the second end of the tubular member rests on a bottom surface of the closed end of the well.

[0013] In one aspect, the cell concentrating device can further include at least one guide member that facilitates positioning of the cell concentrating device in the interior volume of the well. In one embodiment, the at least one guide member can comprise an alignment fin that extends from an exterior side of the tubular member. For instance, the cell concentrating device can include a plurality of alignment fins that are spaced around the circumference of the tubular member. In one aspect, for instance, the cell concentrating device can include from about 4 to about 16 alignment fins.

[0014] Alternatively, the cell concentrating device can include a top member configured to contact and rest on a top surface of the well. The guide member, in this embodiment, can comprise a retention tab that extends downwardly from the top member and is configured to be inserted into the interior volume of the well. In one embodiment, the cell concentrating device can include a combination of alignment fins and retention tabs. In another aspect, one or more alignment fins can be increased in size so that they function as retention elements by pressing against the wall of the well.

[0015] In one aspect, the well plate can comprise a frame and can include a plurality of wells that extend from the frame. The cell concentrating device can also comprise a frame and a plurality of tubular members can extend from the frame of the cell concentrating device. The well plate, for instance, can contain the same or more wells than the number of tubular members on the cell concentrating device. The cell concentrating device can be insertable in the well plate such that for each tubular member on the cell concentrating device, there is a corresponding well. In one aspect, for instance, multiple cell concentrating devices can be used to ensure that there is a tubular member for each well on the well plate. In one embodiment, the cell concentrating device, for instance, can contain from about 8 to about 384 tubular members connected to the frame.

[0016] When using centrifugation to settle cells within the wells, the tubular members on the cell concentrating device can have a unique shape or geometry for providing cell placement in the center of the wells when subjected to centrifugal forces. For instance, in one embodiment, the tubular members can be arranged in rows and each tubular member can have an axis that intersects with a center point of the first open end and a center point of the second open end. The axis of thetubular members in a middle of each row can be vertical (e.g. 90° to the horizontal). The tubular members on an end of each row can have an oblique shape such that the axis is angled with respect to the axis of the middle tubular member. For instance, the axis on the end tubular members can angle away from the axis of the middle tubular member. The axis of the end tubular members, for instance, can form an angle with the axis of the middle tubular member of from about 3° to about 80°, such as from about 5° to about 30°.

[0017] The present disclosure is also directed to a method for seeding cells in a well plate. The method includes adding a liquid medium, such as a buffer, into the open end of each well on the well plate. The cell concentrating device as described above is then brought into engagement with the well plate such that a tubular member is inserted into each of the wells on the well plate after the liquid medium has been loaded into the well. A cell population is then added into each well through the tubular member and allowed to settle on a bottom surface of the well. In this manner, the cells become densified on the bottom of the well where the second end of the tubular member is located.

[0018] In one aspect, the well plate and cell positioning device are centrifuged after the two components have been brought together and the cells have been loaded in the wells. After the cells have settled on a bottom surface of the well, the method can further include the step of removing the cell concentrating device from the well plate. The method can further include the step of obtaining an optical measurement of the cells in the wells in order to analyze the cells.

[0019] The present disclosure is also directed to a cell concentrating device. The cell concentrating device can comprise a plurality of tubular members that extend from a frame. Each tubular member can have a first open end defining a first opening opposite a second open end defining a second opening. The tubular member can taper from the first open end to the second open end such that the first opening is larger than the second opening. The cell concentrating device can be made from non-elastomeric materials and / or from elastomeric materials.

[0020] Each tubular member on the cell concentrating device can have a shape configured to be inserted through the open end of a well. In one aspect, the length of each tubular member can be at least about 90% of the depth of a corresponding well. In one aspect, the tubular member can have a length such that, wheninserted in a well, the distal end does not make contact with the bottom surface of the well. Instead, a gap is formed between the second open end of the tubular member and the bottom surface of the well. The gap can have a length or thickness of from about 5 microns to about 1 ,000 microns. Optionally, at least some of the tubular members contained on the cell concentrating device can include an alignment fin for facilitating positioning of the tubular member within a well.

[0021] In one aspect, the tubular member can include at least two open ends opposite at least two corresponding open distal ends. In this manner, two different populations of cells can be inserted into the well for forming two different concentrated cell locations on the bottom surface of the well.

[0022] Other features and aspects of the present disclosure are discussed in greater detail below.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] A full and enabling disclosure of the present disclosure is set forth more particularly in the remainder of the specification, including reference to the accompanying figures, in which:Figure 1 is a cross-sectional view of one embodiment of a system for analyzing samples in accordance with the present disclosure;Figure 2 is a perspective view of a well plate that may be used in conjunction with the cell concentrating device of the present disclosure;Figure 3 is a perspective view of one embodiment of a cell concentrating device in an inverted position;Figure 4 is a cross-sectional view of a cell concentrating device in accordance with the present disclosure disposed in a well;Figure 5 is a cross-sectional view of a cell concentrating device in accordance with the present disclosure disposed in a well;Figure 6 is another embodiment of a cross-sectional view of a cell concentrating device in accordance with the present disclosure disposed in a well;Figure 7 is another embodiment of a cross-sectional view of a cell concentrating device in accordance with the present disclosure disposed in a well;Figures 8A-8D illustrate one method of using the cell concentrating device as illustrated in Figure 4;Figure 9 is a cross-sectional view of still another embodiment of a cell concentrating device made in accordance with the present disclosure;Figure 10 is a cross-sectional view of another embodiment of a cell concentrating device made in accordance with the present disclosure placed in association with a well;Figure 11 is a cross-sectional view of still another embodiment of a cell concentrating device in accordance with the present disclosure positioned in a well and including a top cover for positioning a pipette;Figures 12A-12D are plan views of results obtained in the example below;Figures 13A-13D are plan views of results obtained in the example below;Figure 14 is a graphical representation of some of the results obtained in the example below.

[0024] Repeat use of reference characters in the present specification and drawings is intended to represent the same or analogous features or elements of the present invention.DETAILED DESCRIPTION

[0025] It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only and is not intended as limiting the broader aspects of the present disclosure.

[0026] In general, the present disclosure is directed to a system and method for positioning cells within a well of a well plate in order to conduct tests on the cells. For instance, in one embodiment, the method and system of the present disclosure can be used in conjunction with an extracellular flux analyzer, such as an Agilent SEAHORSE XF analyzer. The method and system, for instance, can be used to measure the oxygen consumption rate, the proton efflux rate, the extracellular acidification rate, or other metabolic processes that are occurring within the cells after the cells have been seeded in the well. It should be understood, however, that the method and system of the present disclosure can be used with all different types of cell analyzers.

[0027] More particularly, the present disclosure is directed to a cell concentrating device that can be used in conjunction with a well on a well plate forpositioning and concentrating cells on a surface of the well. The cell concentrating device of the present disclosure, for instance, can include one or more tubular members having a first open end opposite a second open end and, in one aspect, can taper from the first open end to the second open end. The tubular member can have a shape configured to be inserted into a well. Once inserted into a well, cells can be fed through the tubular member for directing the cells onto a particular location on the bottom surface of the well.

[0028] The cell concentrating devices of the present technology enable cells to be seeded into selected areas within a larger well using standard pipets and techniques. In some embodiments, the cell concentrating devices restrict the seeded cells to the central well area during incubation. The concentrating devices of the present technology can be used for seeding adherent or suspension cells.

[0029] In some embodiments, the wells of the well plate may be coated with a substance that promotes cell adherence in order to facilitate adherence of suspension cells or improve adherence of adherent cells to the well surface. For example, polycationic coatings, e.g. poly-d-lysine, may be used to promote adherence of any cell type to the surface of the well. In other embodiments, uncoated well plates may be used.

[0030] In one aspect, the second open end of the cell concentrating device has a relatively small diameter. For instance, the second open end of the cell concentrating device can be smaller than the diameter of the well. In this manner, the cell concentrating device can be used for seeding cells in the center of the well and not on the edges of the well adjacent to the walls of the well.

[0031] In one embodiment, the cell concentrating device can include a plurality of tubular members that are arranged in a cluster and have an overall shape configured to be inserted into a single well. In this way, different cell populations can be inserted into the tubular members for placing the cells on the bottom surface of a well at different locations. For instance, different cell types can be placed on the bottom surface of the well in well-defined quantities to, for instance, mimic animal tissues. Positioning the cells at a particular location on the bottom surface of the well can also facilitate preparing cell ensembles, such as spheroid and organoid formation.

[0032] In addition to placing the cells at a particular location on the bottomsurface of the well, the cell concentrating device can also produce densified cell populations that provide various advantages and benefits. For instance, concentrating the cells at a particular location on the bottom surface of the well can facilitate testing of the cells. When conducting light measurements on the cells in conjunction with a sensor, for instance, concentrating the cells at a particular location can produce a stronger, more uniform, and more reliable signal. Positioning of concentrated cells also enhances imaging-based analysis by ensuring cell-cell interactions with less cells and lowering the optical field-of-view for acquiring an image

[0033] A biological sample or cellular material deposited onto the bottom surface of a well in accordance with the present disclosure, for instance, can be analyzed using an apparatus for measuring extracellular flux as shown in FIG. 1. Referring to FIG. 1, for instance, a system 10 is designed to receive a well plate and can be configured to conduct multiple assays simultaneously on biological samples, such as cellular material, contained in the wells. The system 10 as shown in FIG. 1, for instance, can be used to test one or more constituents or cell parameters in each sample. It should be understood, however, that the system 10 is for exemplary purposes only and not intended in any way to limit the scope of the present disclosure.

[0034] The system 10 can be placed in association with a well plate 12 that defines a plurality of sample staging sites or wells for receiving biological samples in accordance with the present disclosure. In one aspect, the well plate 12 can be designed to be placed in association with a plurality of light probes 14. Each light probe 14 is configured to be inserted into a cartridge 16 that is designed to house the light probes 14 and guide the light probes 14 into corresponding wells on the well plate 12. In one aspect, the light probes 14 can be contained within a mounting block 18. The mounting block 18 and the cartridge 16 can be placed in association with a motor for reciprocating back and forth for inserting and removing the light probes and corresponding cartridges into and out of the wells.

[0035] When inserted into a well, the light probe and cartridge can form an assay microchamber volume which enables increased assay sensitivity. For example, in one aspect, a sensor can be placed on the cartridge which is then inserted into a medium in the wells containing the cells. Excitation light can beemitted by the light probe 14 that, in one aspect, can be directed onto the sensor while the sensor is in contact with the fluid medium containing the cells. A nonimaging emission signal is then produced and is sensed by the light probe 14 for making measurements, such as metabolic measurements. For instance, the system can be used for interrogating one or more cellular functions.

[0036] As used herein, a non-imaging emission signal in the context of analyzing cellular samples and / or measuring metabolic processes refers to signals that are not derived from imaging techniques such as microscopy, such as confocal microscopy. Instead, they can stem from methods that assess metabolic activity or biomolecular interactions indirectly, often through biochemical assays or spectroscopic measurements.

[0037] Non-limiting examples of non-imaging emission signals include spectrophotometric assays that can measure changes in the absorbance or emission of light by molecules within a sample; fluorescence-based assays that can make measurements of a fluorescence emission from a solution containing labeled molecules or cellular material, indicating metabolic activity or molecular interactions; luminescence assays that detect the emission of light resulting from chemical reactions or energy transfer processes; and / or radioactive assays in which metabolic processes can be measured using radioactive isotopes as tracers. These non-imaging techniques can provide valuable information about cellular metabolism and biochemical processes without the need for complex imaging setups.

[0038] As shown in FIG. 1 , the system can include one or more processors 20 that can be placed in communication with each of the light probes 14 for receiving and analyzing signals. The signals can be delivered and received using optical fibers.

[0039] The one or more processors 20 can include, for instance, any suitable processing device, such as one or more microprocessors, integrated circuits (e.g., application specific integrated circuits), CPUs, GPUS, field programmable gate arrays, etc. that perform operations. In some embodiments, the one or more processors 20 can be configured to execute computer-readable instructions stored in one or more memory devices to perform operations, such as any of the operations for determining a response phase, a fluorescent intensity or the like.The one or more memory devices can be any suitable media for storing computer- readable instructions and data. For instance, the one or more memory devices can include random access memory such as dynamic random access memory (DRAM), static memory (SRAM) or other volatile memory. In addition, and / or in the alternative, the one or more memory devices can include non-volatile memory, such as ROM, PROM, EEPROM, flash memory, optical storage, magnetic storage, etc.

[0040] The one or more memory devices can store computer-readable instructions that, when executed by the one or more processors 20, cause the one or more processors to perform operations, such as any of the operations implemented by one or more processors described herein. The instructions can be software written in any suitable programming language or can be implemented in hardware.

[0041] The system illustrated in FIG. 1 is for exemplary purposes only. The cell concentrating device of the present disclosure can be used with all different types of cell analyzers. For instance, wells seeded in accordance with the present disclosure can also be used with cell imaging techniques such as microscopy, including fluorescence microscopy.

[0042] As described above, the present disclosure is directed to a cell concentrating device that is designed to be used in conjunction with the well plate 12 as shown in FIG. 1 for concentrating the cells at a particular location on the bottom surface of a well for increasing the accuracy of measurements taken by the system 10 and / or for improving sensitivity. Referring to FIG. 2, one embodiment of a well plate 12 that may be used in accordance with the present disclosure is shown. The well plate 12 includes a frame 24 that holds a plurality of wells 26. The frame 24 can optionally include a top surface and side walls as shown in FIG. 2. Each well 26 includes an open end opposite a closed end. In between the open end and the closed end is at least one side wall. In the embodiment illustrated in FIG. 2, the well plate 12 includes 96 wells. The well plate 12, however, can include anywhere from about 8 wells to about 1 ,536 wells.

[0043] Referring to FIG. 3, one embodiment of a cell concentrating device 28 made in accordance with the present disclosure is shown. The cell concentrating device 28 includes a frame 30. Extending from the frame 30 are a plurality oftubular members 32. The tubular members 32 include a first open end 34 opposite a second open end 38. The first open end 34 defines a first opening while the second open end 36 defines a second open end 38.

[0044] The tubular members 32 of the cell concentrating device 28 have a shape that is configured to be inserted into the wells 26 of the well plate 12. For instance, in the embodiment illustrated in FIG. 3, the cell concentrating device includes 8 tubular members. In one embodiment, the 8 tubular members can be positioned so as to fit across one of the rows of the wells 26 of the well plate 12. In other embodiments, however, the cell concentrating device 28 can include more or less tubular members 32. In one embodiment, for instance, the cell concentrating device 28 can include the same number of tubular members 32 as there are wells 26 in the well plate 12. For instance, the cell concentrating device 28 can contain anywhere from 1 to about 1 ,536 tubular members 32, such as from about 4 tubular members to about 384 tubular members 32, such as from about 8 tubular members to about 192 tubular members. For instance, in various embodiments, the cell concentrating device may include 8 tubular members, 12 tubular members, 24 tubular members, 48 tubular members, or 96 tubular members.

[0045] Referring to FIG. 4, a cross-sectional view of one embodiment of a cell concentrating device 28 inserted into a well 26 of a well plate 12 is illustrated. As shown, the well 26 includes a side wall 42 and a bottom wall 44 that defines a bottom surface 46. The cell concentrating device 28 defines a tubular member 32 that includes a first open end 34 defining a first opening 40 opposite a second open end 36 that defines a second opening 38. The first opening 40 can be larger than the second opening 38 of the tubular member 32. In the embodiment illustrated in FIG. 4, the tubular member tapers from the first open end 34 to the second open end 36, forming a funnel-like shape.

[0046] The frame 30 of the cell concentrating device 28 can include a top member 48 that, in one embodiment, can be configured to rest on a top surface of the well 26 or the well plate 12.

[0047] In order to ensure that the tubular members 32 are positioned inside the well 26 in the proper location, the cell concentrating device 28 can include one or more guide members. In the embodiment illustrated in FIG. 4, for instance, the guide members comprise alignment fins 50. As shown, the alignment fins 50extend from an exterior surface of the side wall of the tubular member 32. The alignment fins 50 help position the second opening 38 at the desired location of the bottom surface 46 of the well 26.

[0048] The tubular member 32 can include a single alignment fin 50 or can include a plurality of alignment fins. For instance, as shown in FIG. 3, each tubular member 32 includes 8 alignment fins 50 spaced around the circumference of the tubular member.

[0049] Referring to FIG. 5, an alternative embodiment of a cell concentrating device 28 is shown. Like reference numerals have been used to indicate similar elements. Similar to FIG. 4, the cell concentrating device 28 in FIG. 5 includes a tubular member 32 extending from a frame 30. The tubular member 32 defines a first open end 34 defining a first opening 40 opposite a second open end 36 defining a second opening 38. The tubular member 32 is shown inserted into a well 26.

[0050] In the embodiment illustrated in FIG. 5, the guide members associated with the cell concentrating device 28 comprise retention tabs 52 instead of the alignment fins 50 as shown in FIG. 4. As illustrated, the retention tabs 52 extend from the top member 48 or the frame 30. More particularly, the retention tabs 52 extend downwardly from the top member 48 and become inserted into the well 26. Once inserted into the well 26, the retention tabs 52 serve to guide the tubular member 32 into position.

[0051] The retention tabs 52 are also shown in FIG. 3. In the embodiment illustrated in FIG. 3, the retention tabs 52 are only located surrounding the first tubular member 32 and the last tubular member 32 in the row of tubular members. By aligning the first and last tubular members 32 of a row in a corresponding plurality of wells 26, the middle tubular members automatically remain in proper alignment.

[0052] In the embodiment illustrated in FIG. 3, there are four retention tabs 52 surrounding the tubular member 32. The cell concentrating device 28, however, can include more or less retention tabs 52. In one embodiment, only a single retention tab 52 is present on the bottom surface of the frame 30 and can be continuous in the form of a ring that matches the inside circumference of the well 26.

[0053] As shown in FIGS. 4 and 5, once the cell concentrating device 28 is engaged with the well 26, the tubular member 32 extends into the well but, in one embodiment, does not contact the bottom surface 46 of the well 26. Instead, a gap 54 remains. For instance, the tubular member 32 as shown in FIGS. 4 and 5 can have a length that is about 90% of the depth of the well 26. For instance, the length of the tubular member 32 can be from about 90% to about 99.9% of the depth of the well 26. In various embodiments, for instance, the length of the tubular member 32 can be greater than about 92%, such as greater than about 94%, such as greater than about 96%, such as greater than about 98%, and less than about 99.5%, such as less than about 99% of the depth of the well. In this manner, the gap 54 is formed between the second open end 36 of the tubular member 32 and the bottom surface 46 of the well 26.

[0054] During seeding of cells into the well, the cells are inserted into the well 26 through the first open end 34 of the cell concentrating device 28. The cells travel downwardly through the second open end 36 of the tubular member 32 and deposit on the bottom surface 46 of the well 26. It was discovered that creating a gap between the second open end 36 of the tubular member 32 and the bottom surface 46 does not cause the cells to disperse over the bottom surface of the well but remain concentrated where the second opening 38 is located. In particular, it was discovered that a fluid-tight seal does not need to be formed between the bottom surface 46 and the second open end 36 of the tubular member 32 for the cells to be seeded at a concentrated location.

[0055] The gap 54 between the bottom surface 46 and the second open end 36 of the tubular member 32 can generally be from about 5 microns to about 1 ,000 microns in length. For instance, the gap 54 can be greater than about 10 microns, such as greater than about 20 microns, such as greater than about 50 microns, such as greater than about 70 microns, such as greater than about 100 microns, and less than about 900 microns, such as less than about 800 microns, such as less than about 700 microns, such as less than about 600 microns, such as less than about 500 microns, such as less than about 400 microns, such as less than about 300 microns.

[0056] Forming the gap 54 as shown in FIGS. 4 and 5 can provide various advantages and benefits. For instance, once cells are seeded in the well 26 in aconcentrated manner, the cell concentrating device 28 is removed from the well 26 and from the well plate 12 prior to conducting any tests or analysis on the cells. Providing the gap 54 allows for the cell concentrating device 28 to be removed from the wells without disturbing the cells.

[0057] In other embodiments, however, the second open end 36 of the tubular member 32 can rest on the bottom surface 46 of the well 26 as long as the tubular member does not form a seal with the bottom of the well that, later, will cause the cell disturbance when removed. For instance, referring to FIG. 6, another embodiment of a cell concentrating device 28 is shown inserted into a well 26.Like reference numerals have been used to indicate similar elements. As shown in the embodiment in FIG. 6, the tubular member 32 of the cell concentrating device 28 has a length such that the second open end 36 rests on the bottom surface 46 of the well 26. In one aspect, for instance, the tubular member 32 and the cell concentrating device 28 can be made from a material that will not form a seal with the bottom surface 46. For instance, in one embodiment, the cell concentrating device 28 can be made from non-elastomeric materials.

[0058] In one aspect, for instance, the cell concentrating device 28 can be made entirely from a thermoplastic polymer material. The cell concentrating device 28 can comprise a single integral structure made from a polymer material through a molding process, such as through injection molding. The polymer material used to form the cell concentrating device 28, for instance, can comprise a material that produces a relatively low friction surface at least along the second open end 36. In one aspect, for instance, the cell concentrating device 28 can be made from a polycarbonate polymer, polypropylene polymer, polymethylmethacrylate polymer, or a polyethylene polymer. In one embodiment, the cell concentrating device 28 is made from a high density polyethylene polymer that not only has low friction characteristics but is completely non-reactive when contacted with living cells.

[0059] Referring to FIG. 7, still another embodiment of a cell concentrating device 28 is shown inserted into a well 26. In the embodiment illustrated in FIG. 7, the second open end 36 of the tubular member 32 contacts and rests on the bottom surface 46 of the well 26. In this embodiment, however, the well 26 defines a raised area 54 designed to contact the second open end 36 of the tubularmember 32. In one embodiment, for instance, the raised area 54 can comprise a circular ring that has a circumference that matches the circumference of the second open end 36 of the tubular member 32. The raised area 54 can also serve to facilitate positioning and location of the cells once deposited into the tubular member 32.

[0060] Referring to FIG. 8, one method for using the cell concentrating device 28 of the present disclosure is illustrated. Like reference numerals have been used to indicate similar elements.

[0061] Referring to FIG. 8A, the cell concentrating device 28 of the present disclosure is shown inserted into a well 26. The method includes first filling the well with a liquid medium. The liquid medium can be placed in the well using a pipette 60 if desired. The liquid medium can comprise, for instance, a buffer. In one aspect, the buffer is fed to the well 26 after the cell concentrating device 28 has been inserted into the well. Alternatively, the buffer can be placed into the well 26 first followed by placement of the cell concentrating device 28.

[0062] After the liquid medium 58 has been fed to the well 26, a pipette 62 can be used to deliver a cell population 56 to the well 26. For instance, as shown in FIG. 8B, the cell population 56 can be fed through the first open end 34 of the cell concentrating device 28. As shown, the shape of the tubular member 32 of the cell concentrating device 28 directs the cells 56 to the bottom surface 46 of the well 26 where the second open end 36 is positioned.

[0063] As shown in FIG. 8C, the cells 56 are then allowed to settle on the bottom surface 46 of the well 26 opposite the second opening 38 of the tubular member 32. The cells 56 can settle by gravity or the well plate 12 can be spun in a centrifuge such that the cells are spun onto the bottom surface 46 of the well 26.

[0064] After the cells 56 have settled, the cell concentrating device 28 can be removed without disturbing the cells as shown in FIG. 8D. In one embodiment, the cell concentrating device 28 can be removed as soon as the cells are deposited on the bottom surface 46 of the well 26. Alternatively, the well plate 12 in combination with the cell concentrating device 28 can be placed in an incubator in order to promote cell growth and proliferation.

[0065] As shown in FIGS. 8A-8D, the cells 56 deposit on the bottom surface 46 of the well 26 where the second opening 38 is located. In one aspect, the cells 56adhere to the bottom surface 46 of the well 26. For instance, the cells can be adherent cells that naturally adhere to the surface. Alternatively, the bottom surface 46 can be suitably prepared to promote cell adhesion. For instance, a coating can be applied to the bottom surface 46 for promoting adhesion including, for example, Corning CELL-TAC coating or a poly-d-lysine coating.

[0066] The cells 56 can also mimic the shape or circumference of the second opening 38 of the tubular member 32. In one aspect, the second opening is circular for forming a circular pattern of cells on the bottom surface. The size or diameter of the second opening 38 determines and influences the size of the cell population that is adhered to the bottom surface of the well 26. For instance, in one aspect, the second opening 38 can have a diameter of from about 0.25 mm to about 5.5 mm. For instance, the diameter (or effective diameter if not circular) of the second opening 38 can be greater than about 0.5 mm, such as greater than about 0.75 mm, such as greater than about 1 mm, such as greater than about 1 .25 mm, such as greater than about 1 .5 mm, and less than about 5 mm, such as less than about 4.5 mm, such as less than about 4 mm, such as less than about 3.5 mm, such as less than about 3 mm, such as less than about 2.5 mm, such as less than about 2 mm. In one particular embodiment, the second opening 38 can have a diameter of from about 1 mm to about 2 mm. It is understood that the second opening is not limited to circular shapes.

[0067] As described above, the cells 56 as shown in FIGS. 8A-8D can settle to the bottom surface of the well using gravity or using a centrifuge. When using a centrifuge, the well plate 12 can be placed in a centrifuge for accelerating the effects of gravity. When placed in a centrifuge, the forces exerted on each well can vary depending upon the position of the well in the well plate. For instance, the wells in a middle of a row on the well plate will experience centrifugal forces that are similar to gravity in a downward vector. The wells that are not in the middle, and especially the wells on the end of each row, will experience centrifugal forces at an angle. For example, when placing a well plate 12 having 96 wells as shown in FIG. 2, the wells at the end of each row will experience centrifugal forces at an approximately 13° angle relative to the middle well. This angle can increase or decrease depending upon the number of wells in each row. Consequently, when using a centrifuge to deposit cells on the bottom surface of the well, thelocation of the cells can vary depending upon the position of the well in a row in relation to the centrifuge device. The cell concentrating device 28 of the present disclosure, however, can be used to counteract the effect of the differences in centrifugal forces from well to well for improving the uniformity of cell deposition.

[0068] For instance, referring to FIG. 9, another embodiment of a cell concentrating device 28 is illustrated. Like reference numerals have been used to indicate similar elements. As shown, the cell concentrating device 28 includes a frame 30. A plurality of tubular members 32 extend from the frame 30. The tubular members 32 include a first open end 34 opposite a second open end 36.

[0069] In order to improve the uniformity of cell deposition while using a centrifuge, the cell concentrating device 28 includes a middle tubular member 32 having a regular geometry and end tubular members 32 that have an oblique shape. For instance, as shown, the middle tubular member 32 includes an axis 70 that is completely vertical and perpendicular to the horizontal. By being in the middle of the cell concentrating device 28, a well placed in association with the tubular member 32 will experience centrifugal forces that are consistent with gravity that causes the cells to deposit on the bottom surface of the well in a downward direction.

[0070] The tubular members 32 positioned at the ends of the row, however, have an oblique shape in order to correct for variations in centrifugal forces when placed in a centrifuge. As shown, the tubular member 32 at one end of the cell concentrating device 28 includes an axis 72 while the tubular member 32 at the opposite end of the cell concentrating device 28 includes an axis 74. The axes 70, 72, and 74 intersect the center point of the first opening at the first open end 34 and intersect the center point of the second opening at the second open end 36. As shown, the axes 72 and 74 angle away from the middle axis 70. The difference in the angles in accordance with the present disclosure is set to match the differences in centrifugal forces when placed in a centrifuge. For instance, the axis 72 and the axis 74 can form an angle with the axis 70 that is from about 2° to about 45°. The angle, for instance, can be greater than about 5°, such as greater than about 10°, and less than about 40°, such as less than about 35°, such as less than about 30°, such as less than about 25°, such as less than about 20°. For instance, in a standard well plate containing 96 wells with 8 wells per row, thetubular members 32 at the end of each row may have an axis that forms an angle with an axis of the middle well of about 13°.

[0071] In FIG. 9, only 3 tubular members are illustrated. In other embodiments, the cell concentrating device 28 can include greater than 3 tubular members per row, such as 8 tubular members or 12 tubular members. When a row contains more than 3 tubular members, the angle of the axis of each tubular member can gradually increase from the middle to the ends. For instance, when containing 8 tubular members, the two middle tubular members can have a vertical axis while the tubular members on each end can have an axis that is angled from about 10% to about 15% in relation to the vertical axis of the middle tubular members. The tubular members positioned between the middle tubular members and the end tubular members can have axes that display gradually increasing angles. The angle of each axis of each tubular member, for instance, can be matched to the centrifugal forces produced by the centrifuge.

[0072] In the embodiments illustrated in FIGS. 3-9, the cell concentrating device 28 includes a single tubular member 32 that corresponds with a single well on a well plate. Alternatively, however, the cell concentrating device 28 can include tubular members that have two or more entry points and two or more exit ports or open ends. In particular, a cluster of tubular members can be arranged together for insertion into a single well on a well plate. Having multiple tubular members inserted into a single well can allow for different populations of cells to be deposited at different locations on the bottom surface of the well. In this embodiment, the same type of cells can be placed at different locations along the bottom of the well or different cell types can be arranged at different locations in the same well. For instance, two different cell populations can be positioned adjacent to each other on the bottom surface of the well in order to observe and measure cell interactions and other phenomenon. In one application, different cell types can be placed at different locations at the bottom of the well in controlled quantities to mimic animal tissues.

[0073] The cell concentrating device of the present disclosure is also well suited for facilitating spheroid formation and for positioning proliferating cells at the bottom of a well. For instance, the cell concentrating device of the present disclosure is well suited for containing proliferating cells within a controlleddiameter at the bottom of the well. In addition, the cell concentrating device of the present disclosure is also well suited for high sensitivity live cell imaging in which cells are deposited in a glass-well plate. In this embodiment, a plate imager can be used to monitor the cells over time.

[0074] The cell concentrating device of the present disclosure can not only be used to position and concentrate cells on the bottom of the well, but can also be used, in one embodiment, to direct and position a pipette within the well for depositing the cells in the well. The performance of the cell concentrating device can be enhanced by positioning the tip of the pipette near the fluid level within it. For instance, FIGS. 10 and 11 represent two further embodiments of cell concentrating devices 28 made in accordance with the present disclosure. Like reference numerals have been used to indicate similar elements.

[0075] Referring to FIG. 10, the cell concentrating device 28 is shown inserted in a well 26. The cell concentrating device 28 includes a tubular member 32 that includes a first open end 34 opposite a second open end 36. The second open end 36 is positioned adjacent to a bottom surface 46 of the well 26.

[0076] As shown in FIG. 10, a pipette 80 is being used to insert a cell population into the well 26 through the tubular member 32. When depositing cells into the well, the leading tip of the pipette 80 should be inserted into the fluid in the well but should not contact the bottom surface 46 of the well 26. Instead, the tip of the pipette 80 ideally is inserted into the well 26 until the tip is a certain distance from the bottom surface 46 of the well 26. For instance, as shown in FIG. 10, a line 84 is included where the tip is ideally located for depositing the cells in the well. The line 84, for instance, can be greater than about 1 mm, such as greater than about 2 mm, such as greater than about 2.5 mm, and less than about 6 mm, such as less than about 5 mm, such as less than about 4 mm, such as less than about 3.5 mm from the bottom surface 46. In this regard, the cell concentrating device 28 can include a locator notch 82. The locator notch 82 can be located along the line 84. When injecting cells into the well, the tip of the pipette 80 can be dragged across the interior wall of the tubular members 32 until the notch 82 is engaged. Once the tip of the pipette 80 engages the notch 82, the cells can be released from the pipette and into the well. The notch 82 can be positioned along one side of the tubular member 32 or can comprise a ring that goes around theentire interior surface of the tubular member

[0077] Referring to FIG. 11 , another embodiment of a cell concentrating device 28 is shown in conjunction with a pipette positioning system. Similar to FIG. 10, the cell concentrating device 28 includes a tubular member 32 inserted into a well 26. The tubular member 32 includes a second open end 36 that is positioned adjacent to a bottom surface 46 of the well 26. In this embodiment, however, instead of a notch, the cell concentrating device 28 includes a cover 86 that defines an aperture 88 for receiving the pipette 80. In this embodiment, the aperture 88 has a size that receives the tip of the pipette 80 and permits a portion of the pipette 80 to enter the well 26 until further movement is stopped by the size of the aperture 88. For instance, in one embodiment, the pipette 80 can have a tapered size such that the circumference of the pipette increases from the tip to at least a middle portion of the pipette. In this embodiment, the aperture 88 can be large enough to accept the tip of the pipette but prevents further movement of the pipette 80 in a downward motion once the diameter of the pipette matches the diameter of the aperture 88. As shown, movement can be stopped at the line 84 where cells can then be deposited into the well 26.

[0078] In other embodiments, the pipette 80 can be equipped with a stop element along the outside circumference of the pipette. The stop element, for instance, can be a ring or tab. In this embodiment, the pipette can be inserted into the aperture 88 until the aperture 88 contacts the stop element.

[0079] In some embodiments, the present methods, apparatus and systems are useful in measuring cell biology, such as in the area of micro-respirometry, which includes quantitatively measuring the bioenergetics or metabolic state of a small number of cells, as opposed to respirometry performed on whole animals. In the past, micro-respirometry was performed with microscopic glass flow cells that utilized milliliters of cell culture and Clark electrodes for measuring cell metabolism. This technique is not microscopic, facile or high-throughput. Flux analyzers and assays from Agilent provided improved technology for micro-respirometry by introducing comprehensive assays that can be easily performed in 8, 24 and 96 plastic cell culture plates. The resulting technology enables complex characterization of both the glycolysis and oxidative phosphorylation pathways, by introducing various stimulants, inhibitors and custom drugs and measuringchanges in oxygen consumption and proton production.

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

[0081] The cell populations used in the present methods and apparatus may include any cells of interest. Such cells include, but are not limited to, bacteria, fungus, yeast, a prokaryotic cell, a eukaryotic cell, an animal cell, a human cell, and / or an immortal cell. At least a portion of the cells may be attached to a surface of the vessel. At least a portion of the cells may be suspended in the media. At least a portion of the cells may include living tissue, organoids, spheroids or engineered tissue. In some embodiments, at least a portion of cells are adhered to a closed end or a wall of the wells.

[0082] Known cell lines can be used as a cell type in the present methods, apparatus, and systems. For example, known cell lines that can be used in conjunction with the present technology include, but are not limited to, C8161 , CCRF-CEM, MOLT, mlMCD-3, NHDF, HeLa, HeLa-S3, Huh1 , Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel , PC-3, TF1 , CTLL-2, CIR, Rath, CV1 , RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1 , SEM-K2, WEHI-231 , HB56, TIB55, Jurkat, J45.01 , LRMB, Bcl-1 , BC-3, IC21 , DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1 , COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB / 3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1 , 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721 , 9 L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431 , A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21 , BR 293, BxPC3, C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1 , CHO-K2, CHO-T, CHO Dhfr- / -, COR-L23, COR-L23 / CPR, COR-L23 / 5010, COR-L23 / R23, COS-7, COV-434, CML T1 , CMT, CT26, D17,DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1 , EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1 , KY01 , LNCap, Ma-Me1 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231 , MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR / 0.2R, MONO- MAC 6, MTD-1A, MyEnd, NCI-H69 / CPR, NCI- H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1 , NW-145, OPCN / OPCT cell lines, Peer, PNT-1A / PNT 2, RenCa, RIN-5F, RMA / RMAS, Saos- 2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1 , YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). These or other cell lines can be employed as a first cell type or a second cell type in the present methods and apparatus. In some embodiments, the first cell type is in a cell population taken from a subject (such as a human patient), and the second cell type is a known cell line.

[0083] The present disclosure may be better understood with reference to the following examples.EXAMPLES Example No. 1

[0084] The cell concentrating devices as shown in FIG. 3 were 3D printed. Two different models were fabricated. The first model included an exit port or second opening at the distal end of the tubular member that had an inside diameter of 0.8 mm. The second model was identical but had an exit port diameter of 2 mm. The cell concentrating devices produced included columns in a 96-well format.

[0085] An Agilent XF 96 well plate was coated with poly-d-lysine. Each well was filled with 150 pl of PBS buffer. Human K562 cells in 10 pl with stained nuclei were deposited into the wells using the cell concentrating devices. After one hour, the cell concentrating devices were removed and the well plate was scanned.

[0086] The results using the 0.8 mm cell concentrating device are illustrated in FIGS. 12A-12D. The results illustrating use of the 2 mm cell concentrating device are illustrated in FIGS. 13A-13D. As shown, the bottom of each well included three dimples. The cells were successfully positioned in the center of the bottom surface of the well plate in a circular configuration avoiding the dimples. Thedifference in diameter between the cell concentrating devices is noticeable when comparing FIG. 12 to FIG. 13.Example No. 2

[0087] The cell concentrating device having a 2 mm diameter exit port as described in Example No. 1 was used to concentrate cells on the bottom surface of a well plate. 10,000 cells were deposited on the bottom surface of the well plate. Two standard well plates were then seeded with 40,000 cells and with 10,000 cells without using the cell concentrating device of the present disclosure. The three well plates were then inserted into the Agilent SEAHORSE XF analysis system and basal metabolism was measured. FIG. 14 provides the oxygen consumption rate for the three different groups of wells. As shown in FIG. 14, the 10,000 cells seeded in the well plate using the cell concentrating device produced a significantly higher signal than the well plate seeded with 10,000 cells without using the cell concentrating device. In fact, the signal using only 10,000 cells with the cell concentrating device was even higher than the well plate that was seeded with 40,000 cells. FIG. 14 demonstrates the increased sensitivity when using the cell concentrating device of the present disclosure.

[0088] These and other modifications and variations to the present invention may be practiced 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 interchanged both in whole or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only and is not intended to limit the invention so further described in such appended claims.

Claims

WHAT IS CLAIMED:1 . A cell seeding system comprising: a well plate including at least one well, the well having an open end opposite a closed end and including at least one side wall extending between the open end and the closed end, the well defining an interior volume and a depth for holding a fluid media; and a cell concentrating device insertable and removable from the well, the cell concentrating device being configured to receive a cell population and direct the cell population to the closed end of the well, the cell concentrating device comprising a tubular member having a first open end defining a first opening opposite a second open end defining a second opening, the first opening being larger than the second opening, the tubular member having a shape configured to be inserted through the open end of the well, the tubular member having a length that is at least about 90% of the depth of the well, the cell concentrating device comprising at least one guide member that facilitates positioning of the cell concentrating device in the interior volume of the well.

2. A cell seeding system as defined in claim 1 , wherein the guide member comprises an alignment fin extending from an exterior side of the tubular member.

3. A cell seeding system as defined in claim 2, wherein the cell concentrating device includes a plurality of alignment fins.

4. A cell seeding system as defined in claim 3, wherein the plurality of alignment fins are spaced around the exterior side of the tubular member, the cell concentrating device including from about 4 to about 8 alignment fins.

5. A cell seeding system as defined in any of the preceding claims, wherein the tubular member of the cell concentrating device defines a taper from the first open end to the second open end.

6. A cell seeding system as defined in any of the preceding claims, wherein the second open end of the tubular member of the cell concentrating device has an inner diameter of from about 0.25 mm to about 5.5 mm, such as from about 0.5 mm to about 3.5 mm, such as from about 0.75 mm to about 2.25 mm.

7. A cell seeding system as defined in any of the preceding claims, wherein the cell concentrating device further comprises a top member configured to contact and rest on a top surface of the well.

8. A cell seeding system as defined in claim 7, wherein the guide member comprises a retention tab that extends downwardly from the top member of the cell concentrating device and is configured to be inserted into the interior volume of the well.

9. A cell seeding system as defined in any of the preceding claims, wherein the tubular member has a length such that when inserted into the well, a gap remains between a bottom surface of the closed end of the well and the second open end of the tubular member.

10. A cell seeding system as defined in claim 9, wherein the gap has a length of from about 5 microns to about 1 ,000 microns, such as from about 50 microns to about 500 microns, such as from about 100 microns to about 300 microns.

11. A cell seeding system as defined in any of the preceding claims, wherein the cell concentrating device is formed as a single integral structure made from a thermoplastic polymer material.

12. A cell seeding system as defined in any of the preceding claims, wherein the tubular member has a length such that when inserted into the well, the second end of the tubular member rests on a bottom surface of the closed end of the well.

13. A cell seeding system as defined in any of the preceding claims, wherein the well plate comprises a frame and wherein a plurality of wells extend from the frame, the cell concentrating device also comprising a frame and wherein a plurality of tubular members extend from the frame of the cell concentrating device, and wherein the well plate contains the same or more wells than the number of tubular members on the cell concentrating device, and wherein for each tubular member on the cell concentrating device, the well plate includes a corresponding well.

14. A cell seeding system as defined in claim 13, wherein the cell concentrating device contains from about 8 to about 384 tubular members attached to the frame.

15. A cell seeding system as defined in claim 14, wherein the tubular members are arranged in rows, each tubular member having an axis that intersects a center point of the first open end and a center point of the second open end, and wherein the axis of the tubular members in a middle of each row are vertical, the tubular members on an end of each row having an oblique shape such that the axis is angled with respect to the vertical axis of the middle tubular member.

16. A cell seeding system as defined in any of the preceding claims, wherein the tubular member defines an interior surface and wherein the interior surface comprises a pipette tip locator.

17. A cell seeding system as defined in claim 15, wherein the pipette tip locator comprises a notch or rim formed into a side wall of the tubular member.

18. A cell seeding system as defined in any of claims 1-14, further comprising a cover configured to go over the first open end of the tubular member, the cover defining an aperture configured to receive a pipette tip, and wherein the aperture has a circumference that limits movement of a pipette tip from being inserted into the well for preventing the pipette tip from contacting a bottom surface of the well.

19. A cell seeding system as defined in claim 1 , wherein the cell concentrating device is made from a non-elastomeric material.

20. A method of seeding cells in a well plate comprising: adding a liquid medium into the open end of the well of a cell seeding system as described in any of the preceding claims; inserting the tubular member of the cell concentrating device into the well containing the liquid medium; and adding a cell population into the well through the tubular member.21 . A method as defined in claim 20, wherein, after the cells have settled on a bottom surface of the well, the method further includes the step of removing the cell concentrating device from the well plate.

22. A method as defined in claim 21 , wherein the cells are settled on a bottom surface of the well using centrifugation.

23. A method as defined in any of claims 20-22, wherein the liquid medium comprises a buffer that does not contain any cells.

24. A method as defined in any of claims 20-23, further comprising the step of obtaining an optical measurement of the cells in the well in order to analyze the cells.

25. A cell concentrating device comprising: a frame; and a plurality of tubular members extending from the frame, the tubular members having a first open end defining a first opening opposite a second open end defining a second opening, the first opening being larger than the second opening, each tubular member having a shape configured to be inserted through an open end of a well on a well plate, each tubular member having a length such that, when the tubular member is inserted into a corresponding well, a gap forms between a bottom surface of the well and the second opening of the tubular member.

26. A cell concentrating device as defined in claim 25, wherein the tubular member is tapered from the first open end to the second open end and wherein the tubular member further comprises a plurality of alignment fins extending from an exterior side surface of the tubular member.

27. A cell concentrating device as defined in claim 25 or 26, wherein the second open end of each tubular member has an inner diameter of from about 0.25 mm to about 5.5 mm, such as from about 0.5 mm to about 3.5 mm, such as from about 0.75 mm to about 2.25 mm.

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