Method and apparatus for cell culture well plates

Abstract

The present invention relates generally to methods, devices, and systems for cell culture in vitro. More specifically, the present invention relates to novel multi-well plates and concentrator masks. The present invention also relates to cell seeding and cell assays using the multi-well plates and concentrator masks.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 928,121, filed October 30, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention generally relates to methods, apparatus, and systems for in vitro cell culture. More specifically, the invention relates to novel multiwell plates and concentrator masks. The invention also relates to cell seeding and cell assays using said multiwell plates and concentrator masks. Background Technology

[0004] Multiwell plates are widely used for parallel and / or simultaneous cell measurements, and are available in various well sizes from suppliers such as Agilent Technologies, Sigma-Aldrich, and Thomas Scientific. Multiwell plates for tissue culture are available in 6-well, 12-well, 24-well, 48-well, 96-well, 384-well, and 1536-well sizes. Coated and uncoated plates can be used for adherent cell culture and suspension culture, respectively.

[0005] Multiwell plates are commonly used for measuring cell populations. Typically, when cells are seeded into the wells of a cell culture plate, a cell solution is transferred into the wells, and the cells settle and cover the bottom of the well. The seeded plate is then placed in an incubator to promote cell growth and expansion. This results in cells covering the entire bottom surface of the well. However, seeding cells across the entire bottom surface of the well presents several challenges, many of which stem from the spatial sensitivity associated with numerous in vitro cell assays.

[0006] Therefore, it is generally desirable to concentrate cultured cells in the center of the well to avoid spatial deviations, including but not limited to cell seeding deviations due to thermal inhomogeneity, which are related to optical measurement sensitivity and cell biological effects caused by the edges.

[0007] For measurements with optical readouts, light transduction from the periphery of the aperture is generally less efficient than transduction from the center. This is due to reduced light access near the aperture sidewalls. Furthermore, illumination and / or data collection efficiency is hindered at the edges of the detector's field of view.

[0008] In some cases, plate reader strategies are employed to generate uniform illumination and facilitate the uniform capture of data from the entire well. However, this strategy is both time-consuming and inefficient. Other plate reader strategies measure only from the center of the well to avoid areas of poor optical transduction, but this approach may miss relevant data from cells growing outside the well center.

[0009] Furthermore, when cells are seeded into well plates, the growth and performance of cells in wells at the outer periphery of the plate may differ from those in wells not at the periphery. This phenomenon is commonly referred to as the "edge effect." This edge effect is significant, so it is generally practiced not to seed cells into the peripheral wells of a multi-well plate. Without being bound by theory, the edge effect is thought to be due to differences in the heating of the culture medium in the peripheral wells of the plate when the plate is placed in an incubator. For example, in response to a thermal gradient, cells tend to aggregate at the well sidewalls. For the wells at the periphery of the plate, the thermal gradient is more pronounced, leading to disproportionate cell proliferation towards the well sidewalls. When analyzing cells exhibiting the edge effect using optical assays, it can lead to increased variability between wells. Some well plates contain moat-like grooves, which users fill with culture medium to mitigate the edge effect. The culture medium is considered to act as a thermal and humidity buffer for the peripheral wells.

[0010] There remains a strong need in the art for devices, methods, and systems for culturing cells in multi-well plates that address the physical and biological challenges associated with cell growth near the sidewalls of the wells and with the optical detection of such cells. Summary of the Invention

[0011] These and other features and advantages of the method and apparatus of the present invention will become apparent from the following detailed description in conjunction with the appended claims.

[0012] In one aspect, the present invention relates to a multi-well plate for cell populations in a liquid culture medium. The multi-well plate includes: a frame having a frame surface and frame sides extending from the frame surface; a plurality of wells, each well having an open end, a closed end opposite the open end, and at least one wall located between the open end and the closed end; wherein the open end of each well is surrounded by the frame surface; wherein the closed end is included between and in contact with the at least one wall on a well surface; and at least one continuous loop on the well surface of the closed end of one or more wells. It is contemplated that the continuous loop can be of any shape, as long as the shape includes a continuous boundary on the closed end of the well. In some embodiments, the continuous loop is circular, elliptical, or other shape including a circular boundary edge. In other embodiments, the continuous loop can be square, rectangular, triangular, or other geometric shapes. In some embodiments, the at least one continuous loop is configured to define at least one cell seeding region on the well surface. The shape of the cell seeding region is defined by the shape of the continuous loop and can be of any shape, as long as it includes a continuous boundary. In some embodiments, the multi-well plate includes one or more continuous rings configured to define one or more cell seeding regions.

[0013] In another aspect, the present invention relates to a concentrator mask for seeding cells in a liquid culture medium into a multiwell plate. The concentrator mask includes: a frame having a frame surface and frame sides extending from the frame surface; a plurality of funnels extending from the frame surface, each funnel having a first open end and a second open end; wherein the first open end is connected to the frame surface and has a larger diameter than the second open end.

[0014] Other aspects of the present invention relate to a method of seeding cells in the central portion of a culture well. The method includes: pipetting a liquid culture medium containing cells into the at least one cell seeding region, the at least one cell seeding region being defined by at least one continuous ring on the surface of the closed end of each well.

[0015] Other aspects of the present invention relate to a cell seeding system comprising a multi-well plate and a concentrator mask. The multi-well plate includes: a frame having a frame surface and frame sidewalls extending from the frame surface; a plurality of holes, each hole having an open end, a closed end opposite the open end, and at least one wall located between the open end and the closed end. The open end of each hole is surrounded by the frame surface; wherein the closed end includes a hole surface between and in contact with the walls; and at least one continuous ring on the hole surface of the closed end of one or more of the holes. The concentrator mask includes: a frame having a frame surface and frame sidewalls extending from the frame surface; and a plurality of funnels extending from the frame surface. Each funnel has a first open end and a second open end, and the first open end is connected to the frame surface and has a larger diameter than the second open end. When the funnel of the concentrator mask is inserted into the plurality of holes, the plurality of funnels of the concentrator mask and the multi-well plate form the cell seeding system such that the second open end of the funnel contacts the continuous ring on the surface of the closed end of each hole.

[0016] Other aspects of the invention relate to a method of seeding cells at the center of a well in a multi-well plate by pipetting a liquid culture medium containing cells into a first open end of a funnel of a cell seeding system, wherein the cells are deposited into an area defined by the at least one continuous ring on the surface of the well. Attached Figure Description

[0017] Figure 1A This is a cross-sectional view of a porous plate having holes including continuous rings on the inner bottom surface of the holes.

[0018] Figure 1B yes Figure 1AAn enlarged view shows a porous plate with holes comprising a continuous ring on the inner bottom surface of the holes.

[0019] Figure 2A This is a schematic diagram of a concentrator mask used to seed cells into a multi-well plate.

[0020] Figure 2B yes Figure 2A A cross-sectional view showing the concentrator mask used for seeding cells into a multi-well plate.

[0021] Figure 3 This is a cross-sectional view of a cell seeding system, which includes a concentrator mask inserted into a porous plate with holes, the holes comprising a continuous ring on the inner bottom surface of the holes.

[0022] Figure 4 This is a schematic diagram of a 96-hole plate, which has holes including a continuous ring on the inner bottom surface of the holes.

[0023] This teaching is best understood from the following detailed description when read in conjunction with the accompanying drawings. These features are not necessarily drawn to scale. In practice, the same reference numerals refer to the same features. Detailed Implementation

[0024] It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive. The defined terms are those beyond their technical and scientific meaning as generally understood and accepted in the technical field of this teaching.

[0025] definition

[0026] As used herein, the term “basically” or “substantially” means, in addition to its ordinary meaning, within the limits or extent acceptable to a person skilled in the art.

[0027] As used herein, the terms “approximately” and “about” mean within limits or quantities acceptable to a person skilled in the art. The term “about” typically refers to plus or minus 15% of the indicated number. For example, “about 10” could indicate a range of 8.5 to 11.5. For example, “approximately the same” means that a person skilled in the art considers the items to be identical after comparison. In this disclosure, numerical ranges include numbers within defined ranges.

[0028] Before describing the various embodiments, it should be understood that the teachings of this disclosure are not limited to the specific embodiments described, and therefore are subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be restrictive, as the scope of this teaching will be defined only by the appended claims.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this teaching, some exemplary methods and materials are described hereafter. All patents and disclosures mentioned herein are expressly combined by reference.

[0030] As used in the specification and appended claims, the terms “a,” “an,” and “the” include both singular and plural references, unless the context clearly specifies otherwise. Thus, for example, “an orifice” includes one or more orifices.

[0031] Porous plate with holes having continuous rings

[0032] In at least one embodiment, the present invention relates to a continuous loop molded to the bottom of each well in a cell culture or assay plate. The continuous loop forms a central cell seeding region at the bottom of the well. When cells are seeded to the center of the loop, the loop acts as a physical barrier, constraining the cells to the center of the well. This has several advantages outlined below.

[0033] Furthermore, when used in conjunction with certain micropore assay instruments (such as Agilent XF instruments), the continuous loop is used to define the volume of the assay microchamber, thereby improving assay sensitivity. For example, in some embodiments, the continuous loop of the pore is mated to the analytical instrument to form a semi-sealed, transient, reduced-volume microchamber in which metabolic measurements can be performed.

[0034] In some embodiments of the present invention, cells are seeded at the center of the well, within the boundary formed by a continuous ring. The continuous ring provides a physical barrier, constraining the seeded cells to the center of the well. Seeding cells only at the center of the well, away from the well sidewalls, mitigates the effects of optical transduction and cell seeding density gradients caused by temperature differences and / or fluid differences near the well periphery. The effects of illumination differences are also minimized because the cells are located away from the well sidewalls.

[0035] In other embodiments, the present invention relates to a method for seeding cells within a continuous loop at the bottom of the wells of a multiwell plate. In some embodiments, cells in a liquid culture medium are added (e.g., pipetted) into a cell seeding area defined by the continuous loop. In these embodiments, the cells can then be expanded, for example, by incubation, but cell expansion is limited to the cell seeding area formed by the continuous loop. The multiwell plates and methods of use described herein are compatible with both adherent and suspension cells. In some other embodiments, cells, tissues, or organoids may be added (e.g., seeded) into a specific area formed by the continuous loop.

[0036] In other embodiments, cells can be seeded both inside and outside a continuous loop, for example, to produce non-contact co-culture of different cell types. In some embodiments, the cell seeding region includes a cell culture medium confined to the cell seeding region by one or more continuous loops. In other embodiments, the cell seeding region includes a cell culture medium in fluid communication with cell culture media from other cell seeding regions.

[0037] In some embodiments, the pores of the porous plate of the present invention may be coated with a substance that promotes cell adhesion, thereby improving the adhesion of suspended cells or promoting the adhesion of adhering cells to the pore surface. For example, a polycationic coating, such as poly-L-lysine, can be used to promote the adhesion of any cell type to the pore surface. In other embodiments, an uncoated porous plate may be used.

[0038] In another embodiment, the present invention relates to the preparation and maintenance of non-contact cell co-culture of different cell types inside or outside a continuous loop. It is also contemplated that the continuous loop used to provide non-contact co-culture can be of any shape, as long as the shape includes a continuous boundary at the closed end of the well. In some embodiments, the continuous loop for non-contact co-culture is circular, elliptical, or other shape including a circular boundary edge. In other embodiments, the continuous loop for non-contact co-culture can be square, rectangular, triangular, or other geometric shapes.

[0039] In some embodiments, at least one continuous ring of any shape is configured to define at least one cell seeding region on the pore surface. In other embodiments, multiple sets of continuous rings (e.g., but not limited to concentric rings) are configured to define more than one cell seeding region on the pore surface. In other embodiments, the molded physical barrier on the pore surface may not be a ring, but rather another configuration configured to provide a grid or cell seeding region.

[0040] When more than one cell seeding region is present on the surface of the well, it is conceivable that each cell seeding region may contain different cell types, the same cell type, a mixture of cell types, or any combination thereof as desired by those skilled in the art. In any case, the shape and number of the cell seeding regions are defined by the shape and number of the continuous rings, and are contemplated to have any shape, size, and / or configuration to provide one or more continuous boundaries to one or more cell seeding regions.

[0041] Further embodiments of the present invention involve using the microplate in conjunction with fiber optic probes, such as those used in Agilent XF instruments. In these embodiments, seeding cells only at the center of the well minimizes optical signal differences caused by radial differential optical signal transduction. This improves both measurement sensitivity and makes signal transduction more uniform throughout the well.

[0042] Unbound by theory, it can be expected that cells confined to the center of the well are less affected by the intrawell thermal gradient that occurs during cell culture workflows and assays. Furthermore, because the cells are confined to the center of the well, they remain at the position of highest optical transduction, thus allowing for increased detection sensitivity and reducing assay variability that may be introduced by cells seeded around the periphery of the well.

[0043] In other embodiments, the wells of the continuous loop form smaller assay microchambers, which achieve greater assay sensitivity using a microplate reader (e.g., an Agilent XF instrument or other well-based assays with radial dependence on signal transduction or on the assay cell seeding area or assay volume). Without being bound by theory, it is also conceivable that cells seeded in the cell seeding region formed by the continuous loop will exhibit reduced edge effects, thereby allowing all wells in the plate to be used for assays with improved well-to-well analysis and homogeneity.

[0044] Figure 1A This is a cross-sectional view illustrating an embodiment of a porous plate 101 of the present invention, the porous plate having holes including continuous rings on the inner bottom surface of the holes. In this embodiment, the porous plate 101 is defined by a frame having a frame surface 102, frame sides 103, and a frame base 104. The frame of this embodiment also includes frame tabs 105 for manipulating the porous plate. The porous plate 101 also includes hole walls 106, open ends 107, and closed ends 108. The closed ends 108 of the holes also include continuous rings 109 defining cell seeding regions 110. In this embodiment, the porous plate 101 also includes peripheral well plate grooves 111, similar to a moat; however, in other embodiments, the porous plate of the present invention does not include peripheral well plate grooves similar to a moat. Figure 1B yes Figure 1A The enlarged view shows an embodiment of the porous plate of the present invention, which has holes comprising a continuous ring on the inner bottom surface of the holes.

[0045] In some embodiments, the cell seeding region surrounded by the continuous ring has an area of ​​about 10% to about 80% of the total pore surface area. In other embodiments, the cell seeding region surrounded by the continuous ring has an area of ​​about 55% to about 75% of the total pore surface area. In some embodiments, the cell seeding region surrounded by the continuous ring has an area of ​​about 60% to about 70% of the total pore surface area. In other embodiments, the cell seeding region is about 65% of the total pore surface area. In other embodiments, the cell seeding region is about 10% to about 25% of the total pore surface area.

[0046] In some embodiments, the continuous ring has a height of about 0.01 mm to about 2 mm. In other embodiments, the continuous ring has a height of about 0.1 mm to about 0.5 mm or about 1 mm. In other embodiments, the continuous ring has a height of about 0.3 mm to about 0.8 mm. In some embodiments, the continuous ring has a height of about 0.2 mm.

[0047] In some embodiments, the continuous ring has an inner diameter of about 0.5 mm to about 6.0 mm. In other embodiments, the continuous ring has an inner diameter of about 1.0 mm to about 5.0 mm. In some embodiments, the continuous ring has an inner diameter of about 3.0 mm to about 4.0 mm. In some embodiments, the continuous ring has an inner diameter of about 2.0 mm.

[0048] The porous plate of this invention can be configured in any manner or orientation, including being configured to have the same number and spacing of holes as a standard porous plate. For example, in some embodiments, the porous plate of this invention includes at least 8 holes. In other embodiments, the porous plate of this invention includes at least 24 holes. In some embodiments, the porous plate of this invention includes at least 48 holes. In other embodiments, the porous plate of this invention includes at least 96 holes. In other embodiments, the porous plate of this invention includes at least 384 holes. In other embodiments, the porous plate of this invention includes at least 1536 holes.

[0049] Concentrator mask for cell seeding

[0050] On the other hand, the present invention provides a cell seeding concentrator mask comprising a plurality of funnels. In some embodiments, the plurality of funnels are configured such that the funnel-shaped orifice inserts are insertable into strips or grids of orifices in a well plate. In some embodiments, the cell seeding concentrator mask is used to constrain cell seeding to a region within the orifice smaller than the bottom of the orifice. In some embodiments, the concentrator mask comprises a frame and a plurality of funnels extending from the frame. In other embodiments, the funnels of the concentrator mask include a first open end and a second open end, wherein the first open end is connected to the frame and has a larger diameter than the second open end of the funnel.

[0051] The cell seeding concentrator mask of this invention allows cells to be seeded into selected areas within larger wells using standard pipetting techniques. In some embodiments, the cell seeding concentrator mask restricts the seeded cells to a central well region during incubation. The concentrator mask of this invention can be used for seeding adherent or suspension cells.

[0052] In some embodiments, the pores of the multiwell plate may be coated with a substance that promotes cell adhesion, thereby improving the adhesion of suspended cells or promoting the adhesion of adherent cells to the pore surface. For example, a polycationic coating, such as poly-L-lysine, can be used to promote the adhesion of any cell type to the pore surface. In other embodiments, an uncoated multiwell plate may be used.

[0053] In some embodiments, the second open end of the plurality of funnels is configured to be smaller than the bottom of the hole into which the funnel is inserted. In these embodiments, the concentrator mask can be used to seed cells at the center of the hole rather than at the edge of the hole near the peripheral wall of the hole.

[0054] In some embodiments, the invention relates to a method in which cells in a liquid solution are pipetted into the first open end of a concentrator mask funnel, which is inserted into the wells of a multiwell plate, and the cells are allowed to settle. In other embodiments, the multiwell plate is rotated in a centrifuge such that the cells are rotated onto an adhesive coating on the bottom of the wells. The multiwell plate can then be moved to an incubator to promote cell growth and expansion. In some embodiments, the concentrator mask remains in the well plate during incubation. In other embodiments, the concentrator mask can be removed from the well plate prior to incubation. In yet another embodiment, the concentrator mask remains in the well plate during incubation and is removed prior to analysis of the seeded cells.

[0055] In another embodiment of the invention, the distal end of the second open end of the funnel forms an interface with the bottom of the pore. In some embodiments, the interface is a liquid-impermeable seal, but in other embodiments, the interface allows but reduces the passage or diffusion of liquid, for example, by providing a gap. In this embodiment, a cell solution is drawn into the first open end of the funnel, and the solution fills the pores inside and outside the second open end of the funnel. Without being bound by theory, it can be envisioned that cells settle to the bottom of the pore by gravity; therefore, the number of cells deposited on the bottom region depends on the number of cells suspended above the bottom of the pore. Therefore, the cell concentrator mask of this embodiment is designed such that it physically occupies the volume above the bottom region of the pore where cells are not desired. Thus, the end result of cell seeding according to this embodiment of the invention is high-concentration cell seeding at the center of the pore, while the peripheral region near the pore wall is unseeded or seeded with low-concentration cells.

[0056] In some embodiments, the outer diameter of the second open end of the funnel is configured to substantially match the inner diameter of the orifice into which it is inserted, such that the inserted funnel is held in place by compression or interference fixation. In some embodiments, the outer diameter is configured to provide a predetermined gap with the inner diameter of the orifice or a portion thereof. The funnel of the present invention can be configured to be inserted into an orifice of any size. Furthermore, for this embodiment, the size of the region in which high concentrations of cells are injected is determined by the inner diameter of the second open end of the funnel and how the funnel is configured to be inserted into the orifice of the multiwell plate.

[0057] In another embodiment, the present invention provides a cell concentrator mask comprising a funnel having a second open end, wherein the second open end includes a distal elastomeric portion. In some embodiments, the distal elastomeric portion of the second open end forms an interface with the bottom of the well, which is a liquid-proof seal. In this embodiment, a cell suspension can be transferred into the first open end of the funnel and into the bottom of the well, thereby displacing the air at the bottom of the funnel with a liquid cell solution. In this embodiment, the cell suspension will seed cells only at the bottom of the well within the inner diameter of the second open end of the funnel. In this embodiment, the seeded cells settle to the bottom of the well with the funnel inserted into the well. The multiwell plate can then be moved into an incubator to promote cell growth and proliferation. In some embodiments, the concentrator mask remains in the well plate during incubation. In other embodiments, the concentrator mask can be removed from the well plate prior to incubation. In another embodiment, the concentrator mask remains in the well plate during incubation and is removed prior to analysis of the seeded cells. In a specific embodiment of the present invention, a concentrated area of ​​cells is inoculated at the center of the pore, while no cells are present around the pore near the edge of the pore wall.

[0058] In some embodiments, the distal elastomeric portion of the second open end comprises a flexible sealing material, such as an elastic, substantially fluid-impermeable material in the form of an O-ring. The flexible sealing material can be any shape adapted to the end of the second open end. For example, the flexible sealing material can be an annular O-ring, a washer with a rectangular cross-section, a metal washer, or other types of flexible materials. In one embodiment, the flexible sealing material can be a fluoropolymer or other material that will form a fluid seal with the opposing orifice surface. In another embodiment, the flexible sealing material is silicone rubber. In some embodiments, the flexible sealing material forms a radial seal between the second open end and the orifice surface. Other sealing orientations are also conceivable. The compliant sealing material can be a variety of rubbers, depending on the temperature and other cell culture medium components and conditions used, such as fluoropolymers, Bunan-N, EPDM, or, in some cases, metals with a flexible outer coating. If the O-ring material permits, the flexible sealing material can also be coated with a chemically inert, biocompatible coating.

[0059] In some embodiments, the concentrator mask of the present invention is configured to mate with a flat-bottomed cell culture well. In other embodiments, the concentrator mask of the present invention is configured to mate with a recessed well (such as, for example, an Agilent XF well).

[0060] In other embodiments, the concentrator mask of the present invention is configured to interface with an in-molecular aperture, the aperture comprising a continuous ring molded into the bottom of the aperture.

[0061] Therefore, the concentrator mask of the present invention can be used in methods of seeding cells in a central cell seeding region within a larger well. In some embodiments, the concentrator mask funnel is used in methods of concentrating cell seeding at the center of the well bottom. In some embodiments, the concentrator mask seeds the majority of cells at a desired cell concentration above the bottom region of the central well. In other embodiments, the concentrator mask of the present invention can be used in methods of excluding cell seeding from the bottom region of the well from which no cells are desired.

[0062] In some embodiments, the cell seeding concentrator mask of the present invention can be used in conjunction with a cell suspension, centrifugation, and surface coating to allow cells in the suspension to adhere to the pore surface. In some embodiments, the concentrator mask can be removed prior to downstream analysis.

[0063] The method of the present invention may further include cell analysis, for example, analysis by Agilent XF assay.

[0064] In some methods of the present invention, the method further includes, for example, removing air trapped at the bottom interface of the concentrator mask / hole by pipetting.

[0065] In some embodiments, approximately 5.0 μl to approximately 20 μl of culture medium may be pipetted into the concentrator mask. In other embodiments, approximately 10 μl to approximately 15 μl of culture medium may be pipetted into the concentrator mask. In some embodiments, approximately 12.5 μl of culture medium may be pipetted into the concentrator mask. Therefore, the method of the present art can be performed using volumes that do not introduce significant pipetting errors.

[0066] In some embodiments of the present invention, the seeded multiwell plate can be moved to an incubator for cell expansion. In some of these embodiments, the concentrator mask can be held in place during cell incubation. Therefore, some embodiments of the present invention are suitable for low-adhesion cells. Other embodiments of the present invention relate to the culture of high-adhesion cells.

[0067] In other embodiments, the concentrator mask and its application method are compatible with commercially available perforated plates (e.g., Agilent XF perforated plates). In yet another embodiment, the concentrator mask and its application method are compatible with current-technology perforated plates including continuous rings on the inner bottom perforated surface.

[0068] Figure 2A This is a schematic diagram of a concentrator mask used for seeding cells into a multiwell plate. In this embodiment, the concentrator mask 201 is defined by a frame surface 202 and frame sides 203. In this embodiment, the frame also includes frame tabs 204 for aligning and stacking the concentrator mask onto the multiwell plate. A plurality of funnels 205 extend from the frame. The funnels include a first open end 206 and a second open end 207, wherein the first open end 206 has a larger diameter than the second open end 207. In some embodiments, the distal end 208 of the funnel includes an elastomeric portion. In some embodiments, the funnel 205 is inserted into a well, wherein a liquid-resistant interface is formed between the bottom of the well and the distal end 208 of the funnel. Figure 2B yes Figure 2A A cross-sectional view showing the concentrator mask used for seeding cells into a multi-well plate.

[0069] The concentrator mask of this invention can be configured in any manner or orientation, including funnels of appropriate number and spacing to be compatible with and fit into a standardized porous plate. For example, in some embodiments, the concentrator mask of this invention includes at least 8 funnels. In other embodiments, the concentrator mask of this invention includes at least 12 funnels. In other embodiments, the concentrator mask of this invention includes at least 24 funnels. In some embodiments, the concentrator mask of this invention includes at least 48 funnels. In other embodiments, the concentrator mask of this invention includes at least 96 funnels. In other embodiments, the concentrator mask of this invention includes at least 384 funnels. In other embodiments, the concentrator mask of this invention includes at least 1536 funnels.

[0070] In some embodiments, the concentrator mask of the present invention is configured to include a funnel having an inner diameter of about 0.5 mm to about 6.0 mm. In some other embodiments, the concentrator mask of the present invention is configured to include a funnel having an inner diameter of about 1.0 mm to about 5.0 mm. In other embodiments, the concentrator mask of the present invention is configured to include a funnel having an inner diameter of about 3.0 mm to about 4.0 mm. In other embodiments, the concentrator mask of the present invention is configured to include a funnel having an inner diameter of about 2.0 mm.

[0071] Cell seeding system including multiwell plates and concentrator masks

[0072] The present invention also relates to a cell seeding system comprising a concentrator mask as described herein, used in conjunction with a continuous annular multiwell plate, also as described herein. In this embodiment, the plurality of funnels of the concentrator mask are configured to fit into the plurality of holes of the multiwell plate.

[0073] The cell seeding system of the present invention includes a multi-well plate, the multi-well plate comprising: a frame having a frame surface and frame sidewalls extending from the frame surface; a plurality of holes, each hole having an open end, a closed end opposite to the open end, and at least one wall located between the open end and the closed end; wherein the open end of each hole is surrounded by the frame surface; wherein the closed end is included between the walls and in contact with the wall on a hole surface; and at least one continuous ring on the hole surface at the closed end of each hole.

[0074] The cell seeding system further includes a concentrator mask comprising a frame having a frame surface and frame sides extending from the frame surface; a plurality of funnels extending from the frame surface, each funnel having a first open end and a second open end; and wherein the first open end is connected to the frame surface and has a larger diameter than the second open end.

[0075] In the cell seeding system of the present invention, the plurality of funnels of the concentrator mask are configured to fit into the plurality of holes of the multiwell plate such that the second open end of the funnel contacts the continuous ring on the surface of the closed end of each hole. In some embodiments, the bottom of the distal end contacts the top of the continuous ring. In some embodiments, the lateral portion of the distal end contacts the lateral portion of the continuous ring.

[0076] Figure 3 This is a cross-sectional view of a cell seeding system, including a concentrator mask inserted into a multi-well plate with holes, the holes including a continuous ring on the surface of the hole at a closed end. In this embodiment, the cell seeding system 301 is formed by a concentrator mask 201 and a multi-well plate 101, the funnel of the concentrator mask 201 being configured to assemble such that the funnel of the concentrator mask is inserted into the hole of the multi-well plate 101. The cell seeding system 301 includes a porous frame surface 102, porous frame sides 103, and a porous frame base 104. In this embodiment, the porous frame also includes porous frame tabs 105 for manipulating a plate. The multi-well plate includes hole walls 106 (not visible) defining holes, and in this embodiment, peripheral well plate grooves 111 resembling a moat. The multi-well plate holes also include an open end (not visible) and a closed end 108, the closed end including a continuous ring 109. The continuous ring 109 is formed in a cell seeding region 110 on the central portion of the closed end 108.

[0077] The concentrator mask 201 of the cell seeding system 301 includes a frame surface 202 and frame sides 203. The concentrator mask also includes a plurality of funnels 205. Each funnel includes a first open end 206 and a second open end 207. The concentrator mask 201 is configured such that the second open end 207 of the funnel is inserted into a hole in a porous plate 101. In this embodiment, the second open end 207 of the funnel is configured to contact a continuous ring 109 on the surface of the closed end of the hole 108. In some embodiments, the second open end 207 includes a distal portion 208. In some embodiments, the interface between the second open end 207 and the continuous ring 109 forms a liquid-tight seal. In some embodiments, the interface between the second open end 207 and the continuous ring 109 does not form a liquid-tight seal. The cell seeding system also includes a porous plate frame tab 105 for a manipulator plate. The cell seeding system also includes a concentrator mask frame tab 204 for aligning and inserting the concentrator mask 201 into the porous plate 101.

[0078] The present invention also relates to a method of seeding cells into the center of wells in a multiwell plate using a cell seeding system 301. In some embodiments, the method includes adding a liquid culture medium containing cells to a first open funnel end 206 of the cell seeding system, such that cells are deposited into a cell seeding region 110 defined by at least one continuous ring 109 on the surface of the well at the closed end of the well 108. In some embodiments, the seeded cell seeding system 301 can be moved to an incubator for cell expansion. In some embodiments, a concentrator mask 201 can be removed from the multiwell plate 101. In other embodiments, the concentrator mask 201 can be retained inserted in the multiwell plate 201 of the cell seeding system 301. In some embodiments, the concentrator mask 201 is removed from the multiwell plate 101 prior to incubation. In other embodiments, the concentrator mask 201 can be retained inserted in the multiwell plate 101 while cells are incubated for expansion. In some embodiments, the concentrator mask 201 can be removed from the multiwell plate 101 prior to cell analysis. In other embodiments, the concentrator mask 201 may remain in the multiwell plate 101 during cell analysis. In some embodiments, cell analysis includes optical readout.

[0079] The technology of this invention relates to and is applicable to specifications with 6 holes, 12 holes, 24 holes, 48 ​​holes, 96 holes, 384 holes and 1536 holes. Figure 4This is a schematic diagram of a 96-well plate of the present invention, which has holes including continuous rings on the inner bottom surface of the holes. In this embodiment, the multi-well plate 401 is defined by a frame having a frame surface 102, frame sides 103, and a frame base 104. The multi-well plate 401 also includes hole walls 106, open ends 107, and closed ends 108 (not visible). The closed ends 108 of the holes also include continuous rings 109 defining cell seeding areas 110. In this embodiment, the multi-well plate 401 does not include peripheral well plate grooves similar to moats or frame tabs for manipulating plates; however, in other embodiments, the multi-well plate of the present invention includes peripheral well plate grooves similar to moats or frame tabs for manipulating plates.

[0080] Metabolic measurement

[0081] In some embodiments, the methods, apparatus, and systems of the present invention can be used to measure cell biology, such as in the field of microrespiration assays, which involve quantitatively measuring the bioenergetic or metabolic state of a small number of cells, rather than performing respiration assays on the whole animal. Historically, microrespiration assays were performed using microscopic glass flow cells, which utilize milliliters of cell culture and Clark electrodes to measure cellular metabolism. This technique is neither microscopic, readily available, nor high-throughput. Seahorse Bioscience's throughput analyzers and assays provide an improved technique for microrespiration assays by introducing comprehensive assays that can be easily performed in 8, 24, and 96 plastic cell culture plates. The resulting sophisticated characterization of glycolysis and oxidative phosphorylation pathways is achieved by introducing a variety of stimulants, inhibitors, and custom-made drugs and measuring changes in oxygen consumption and proton production. Additional details relating to microrespiration assays are provided in U.S. Patent Application No. 15 / 896,255, which is incorporated herein by reference in its entirety.

[0082] As another aspect of the invention, the methods, apparatus, and systems of the invention are used for metabolic measurements of various cell types in culture. In some embodiments, the methods, apparatus, and systems of the invention can be used to analyze cells in non-contact co-culture. The system may include a multi-well plate as described herein. For example, the multi-well plate described herein can be used to maintain non-contact co-culture by placing a first cell type within a cell seeding area defined by a continuous loop and placing a second cell type between the continuous loop and the well walls. In some embodiments, the different cell types in the non-contact co-culture are in fluid communication with each other. In other embodiments, the different cell types in the non-contact co-culture are not in fluid communication with each other.

[0083] Other aspects of the invention include one or more continuous rings of any shape, wherein said continuous rings provide multiple cell seeding regions or segments of any size, shape, or configuration for preparing and maintaining non-contact co-cultures by seeding different cells into different cell seeding regions / segments. Furthermore, certain aspects of the invention include molded physical barriers on the well surfaces, which are not rings but are configured to provide a grid or alternative configuration for cell seeding regions. When non-contact co-cultures of the invention are generated by seeding different cell types into different cell seeding regions, the methods, apparatus, and systems of the invention can be used to independently obtain metabolic measurements from a single cell seeding region / segment at a given time. Optionally, the methods, apparatus, and systems of the invention can be used to obtain metabolic measurements from two or more or all cell seeding regions / segments at a given time. Additional details relating to non-contact co-culture are provided in U.S. Patent Application No. 15 / 896,255, which is incorporated herein by reference in its entirety.

[0084] In the methods, apparatus, and systems of this invention, one or more sensors can be used to measure the physiological characteristics of cell populations. Sensors can be fluorescence sensors, luminescent sensors, ISFET sensors, surface plasmon resonance sensors, sensors based on optical diffraction principles, sensors based on wood anomaly principles, acoustic sensors, or microwave sensors. The techniques of this invention are not limited to any specific cell assay, measurement, or sensor, but can be used by those skilled in the art in combination with any desired cell analysis method. Therefore, the systems, apparatus, and methods of this invention can include one or more of the aforementioned sensors, positioned to measure one or more characteristics of the well sample described herein.

[0085] The methods, apparatus, and systems of this invention can be used in a variety of fields related to cell culture and analysis. These 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 to influence cellular metabolism in co-culture, protein secretion, or intracellular / extracellular ion exchange. The methods, apparatus, and systems of this invention can also be used to determine the health status of cells cultured in vitro (including co-culture) before and after routine assays, thereby improving the performance of such assays.

[0086] cell population

[0087] The cell population used in the methods and apparatus of this invention may include any cells of interest. These cells include, but are not limited to, bacteria, fungi, yeast, prokaryotic cells, eukaryotic cells, animal cells, human cells, and / or immortalized cells. At least a portion of the cells may be attached to the surface of the container. At least a portion of the cells may be suspended in a culture medium. At least a portion of the cells may include living tissue, organoids, spheroids, or engineered tissue. In some embodiments, at least a portion of the cells adheres to the closed end or wall of the pore.

[0088] Known cell lines can be used as cell types in the methods, apparatus and systems of the present invention. For example, known cell lines that can be used in conjunction with the technology of this invention include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, CIR, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, 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, and Hep. G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial cells, BALB / 3T3 mouse embryonic fibroblasts, 3T3 Swiss, 3T3-L1, 132-d5 human embryonic fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, 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, CMLT1, 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, KYO1, LNCap, Ma-Mel 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 lines, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and their transgenic varieties. Cell lines are available from a variety of sources known to those skilled in the art (see, for example, the United States Type Culture Collection (ATCC) (Manassas, Virginia)). In the methods and apparatus of the present invention, these or other cell lines may be used as a first cell type or a second cell type. In some embodiments, the first cell type is a cell population taken from a subject (e.g., a human patient), and the second cell type is a known cell line.

[0089] Exemplary Implementation

[0090] 1. A multi-well plate for cell populations in liquid culture medium, the multi-well plate comprising:

[0091] A frame having a frame surface and frame sides extending from said frame surface;

[0092] A plurality of holes, each hole having an open end, a closed end opposite the open end, and at least one wall located between the open end and the closed end; wherein the open end of each hole is surrounded by the frame surface; wherein the closed end is included between and contacts the at least one wall on the hole surface; and

[0093] At least one continuous ring on the surface of the hole at the closed end of one or more of the holes.

[0094] 2. The multi-well plate according to embodiment 1, wherein the at least one continuous ring is configured to define at least one cell seeding area on the surface of the well, the at least one cell seeding area being able to cooperate with a cap, plunger or another element to form a measurement microchamber.

[0095] 3. The multi-well plate according to embodiment 1, wherein the multi-well plate includes one or more continuous rings configured to define one or more cell seeding regions.

[0096] 4. The perforated plate according to any one of the foregoing embodiments, wherein the at least one continuous ring has a height of about 0.01 mm to about 2 mm.

[0097] 5. The perforated plate according to any one of the foregoing embodiments, wherein the at least one continuous ring has an inner diameter of about 0.5 mm to about 6.0 mm, for example, about 2.0 mm.

[0098] 6. A concentrator mask for seeding cells from a liquid culture medium into a multi-well plate, the concentrator mask comprising:

[0099] A frame having a frame surface and frame sides extending from said frame surface;

[0100] Multiple funnels extending from the surface of the frame, each funnel having a first open end and a second open end;

[0101] The first open end is connected to the surface of the frame and has a larger diameter than the second open end.

[0102] 7. The concentrator mask according to embodiment 6, wherein the second open end includes a distal elastomer portion.

[0103] 8. The concentrator mask according to embodiment 6 or 7, wherein the concentrator mask comprises at least 8 funnels.

[0104] 9. The concentrator mask according to embodiments 6 to 8, wherein the funnel has an inner diameter of about 0.5 mm to about 6.0 mm, for example, about 2.0 mm.

[0105] 10. A method for seeding cells in the central portion of a culture well, the method comprising:

[0106] A liquid culture medium containing cells is added to at least one cell seeding area on the surface of the well according to any one of embodiments 1 to 5.

[0107] 11. The method according to embodiment 10, wherein after cell seeding, the culture well is transferred into an incubator and incubated.

[0108] 12. The method according to embodiment 11, wherein the incubated cells have essentially no or do not exhibit any edge effect caused by the incubator.

[0109] 13. A cell seeding system, comprising a multiwell plate and a concentrator mask:

[0110] The porous plate includes: a frame having a frame surface and frame sides extending from the frame surface; a plurality of holes, each hole having an open end, a closed end opposite to the open end, and at least one wall located between the open end and the closed end; wherein the open end of each hole is surrounded by the frame surface; wherein the closed end is included between the walls and in contact with the wall on a hole surface; and at least one continuous ring on the hole surface at the closed end of each hole.

[0111] The concentrator mask includes a frame having a frame surface and frame sides extending from the frame surface; a plurality of funnels extending from the frame surface, each funnel having a first open end and a second open end; and wherein the first open end is connected to the frame surface and has a larger diameter than the second open end; and

[0112] The plurality of funnels are configured to be fitted into the plurality of holes such that the second open end of the funnel forms an interface with the continuous ring on the hole surface of the closed end of one or more of the holes.

[0113] 14. The system according to embodiment 13, wherein the interface between the second open end and the continuous ring is small enough to reduce the gap for liquid diffusion.

[0114] 15. The system according to embodiment 13, wherein the second open end includes a distal elastomer portion.

[0115] 16. The system according to embodiment 15, wherein the interface is a physical contact between the distal elastomeric portion of the second open end and the continuous ring, thereby forming a liquid-tight seal.

[0116] 17. A method for seeding cells in a multi-well plate, the method comprising:

[0117] A liquid culture medium containing cells is added to the first open end of the cell seeding system according to embodiment 13, wherein the cells are deposited into a region defined by the at least one continuous ring on the surface of the pore.

[0118] 18. The method according to embodiment 17, wherein, after cell seeding, the multiwell plate is transferred to an incubator. The cells are expected to incubate without exhibiting any or substantially no edge effects caused by the incubator.

[0119] 19. The method according to embodiment 18, wherein the concentrator mask is removed from the porous plate before the porous plate is placed into the incubator.

[0120] 20. The method according to embodiment 17 further includes analyzing the cells in the porous plate to obtain optical measurement results.

[0121] Example 1:

[0122] Cells were seeded, amplified, and analyzed in multi-well plates containing continuous loops according to the technique of the present invention. These cells were compared with control cells seeded, amplified, and analyzed in multi-well plates containing standard wells without continuous loops. The continuous loops tested had a wall height of 0.2 mm and an inner diameter of 2.0 mm. The same number of cells (4,500) were analyzed for both well types.

[0123] After cell seeding and expansion, oxygen consumption rate (OCR) was measured using an Agilent XFp instrument. OCR was measured by obtaining optical measurements. Results showed that the same number of cells (4,500) produced a three-fold higher signal intensity when cultured in wells with continuous loops compared to cells cultured in standard wells without continuous loops.

[0124] In view of this disclosure, it should be noted that the methods and apparatus described herein can be implemented in accordance with these teachings. Furthermore, various components, materials, structures, and parameters are included by way of illustration and example only and are not intended to be limiting. In view of this disclosure, these teachings can be implemented in other applications, and components, materials, structures, and devices for implementing these applications can be identified while remaining within the scope of the appended claims.

Claims

1. A multi-well plate for inoculating cell populations in a liquid culture medium, the multi-well plate comprising: A frame having a frame surface and frame sides extending from said frame surface; A plurality of holes, each hole having an open end, a closed end opposite the open end, and at least one wall located between the open end and the closed end; wherein the open end of each hole is surrounded by the frame surface; wherein the closed end is included between and contacts the at least one wall on the hole surface; and At least one continuous ring on the surface of the pore at the closed end of one or more of the pores, wherein the at least one continuous ring is configured to define at least one cell seeding area on the surface of the pore, and wherein the at least one continuous ring provides a physical barrier to constrain the seeded cells to the center of the pore.

2. The porous plate according to claim 1, wherein, The at least one continuous ring has a height of 0.01 mm to 2 mm.

3. The porous plate according to claim 1, wherein, The at least one continuous ring has an inner diameter of 0.5 mm to 6.0 mm.

4. A method for seeding cells in the central portion of a culture well, the method comprising: A liquid culture medium containing cells is added to at least one cell seeding area on the surface of the wells in the multi-well plate according to any one of claims 1-3.

5. The method of claim 4, further comprising incubating the culture wells after cell inoculation.

6. The method of claim 5, wherein the incubated cells do not exhibit edge effects caused by the incubator.

7. A cell seeding system comprising a multi-well plate and a concentrator mask according to any one of claims 1-3: in, The concentrator mask includes a frame having a frame surface and frame sides extending from the frame surface; at least one funnel extending from the frame surface, wherein the at least one funnel has a first open end and a second open end; and wherein the first open end is connected to the frame surface and has a larger diameter than the second open end; and The at least one funnel is configured to be fitted into at least one of the plurality of holes such that the second open end of the funnel forms an interface with the continuous ring on the hole surface of the closed end of one or more of the holes.

8. The system of claim 7, wherein the interface between the second open end and the continuous ring is a gap sufficient to reduce liquid diffusion.

9. The system of claim 7, wherein the second open end includes a distal elastomer portion.

10. The system of claim 9, wherein the interface between the distal elastomeric portion of the second open end and the continuous ring forms a liquid-tight seal.

11. A method for seeding cells in a multi-well plate, the method comprising: A liquid culture medium containing cells is added to the first open end of the cell seeding system according to claim 7, wherein the cells are deposited into a region defined by the at least one continuous ring on the surface of the pore.

12. The method according to claim 11, wherein, After the cells are seeded, the multiwell plate is transferred to an incubator.

13. The method according to claim 12, wherein, Before moving the porous plate into the incubator, the concentrator mask is removed from the porous plate.

14. The method of claim 11, further comprising analyzing the cells in the porous plate to obtain optical measurements.

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