Methods and apparatus for cell culture well plates

Abstract

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

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 928,121, filed on October 30, 2019, the content of which is incorporated herein by reference.

[0002] The present invention generally relates to methods, devices, and systems for cells in culture. More specifically, the present invention relates to novel multi - well plates and concentrator masks. The present invention also relates to the use of multi - well plates and concentrator masks for cell seeding and cell assays.

Background Art

[0003] Multi - well plates are widely used for performing cell measurements in parallel and / or simultaneously and are commercially available in various well formats from vendors such as Agilent Technologies, Sigma - Aldrich, Thomas Scientific. Multi - well plates for tissue culture are available in 6, 12, 24, 48, 96, 384, and 1536 well formats, and coated plates and uncoated plates can be used for adherent cell culture and suspension culture, respectively.

[0004] Multi - well plates are often used for measuring cell populations. Typically, when cells are seeded into the wells of a cell - culture well plate, an aqueous cell solution is pipetted into the wells and the cells settle to cover the bottom of the wells. The seeded well plate is often placed in an incubator to promote cell growth and proliferation. As a result, the cells cover the entire bottom surface of the wells. However, there are several issues arising from seeding cells over the entire bottom surface of the wells, many of which are due to the spatial sensitivity associated with many in vitro cell assays.

[0005] Therefore, it is often desirable to concentrate cultured cells in the center of the well to avoid spatial biases, including but not limited to cell seeding bias dyes, which are associated with optical assay sensitivity and thermal heterogeneity related to edge-induced cell biological effects.

[0006] In assays involving optical readings, the efficiency of light transfer from the peripheral regions of a well is typically lower than that from the center of the well. This is due to reduced light access near the side walls of the well. Furthermore, illumination and / or data acquisition efficiency is hindered at the edges of the detector's field of view.

[0007] In some cases, a plate reader strategy is used to generate uniform illumination and facilitate uniform data acquisition from across the well. However, such strategies are time-consuming and inefficient. Other plate reader strategies perform measurements only from the center of the well to avoid areas with poor light transmission, but this approach may omit data related to cells growing outside the center of the well.

[0008] Furthermore, when cells are seeded into a well plate, cells in wells on the outer perimeter of the plate may grow differently from cells in wells not on the perimeter. This phenomenon is commonly called the "edge effect." Because this edge effect is significant, it is common practice not to seed cells into the perimeter wells of a multi-well plate. Without being constrained by theory, the edge effect is thought to be due to the difference in heating of the well medium in the perimeter wells when the plate is placed in an incubator. For example, cells tend to aggregate towards the side walls of wells in response to temperature gradients. In the case of perimeter wells, the temperature gradient becomes steeper, causing unbalanced cell growth towards the side walls of the wells. When cells exhibiting the edge effect are analyzed using optical assay techniques, there may be greater variability between wells. Some well plates include a moat that the user fills with culture medium to mitigate the edge effect. The medium is thought to act as a buffer for heat and humidity to the surrounding wells.

[0009] There remains a strong need in the art for apparatus, methods, and systems for culturing cells in multiwell plates that address the physical and biological challenges associated with cells growing near the side walls of wells and the optical detection of such cells. [Overview of the project]

[0010] These and other features and advantages of the method and apparatus will become apparent from the following detailed description, in conjunction with the attached claims.

[0011] In one embodiment, the technology relates to a multiwell plate for a cell population in a liquid medium. The multiwell 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 between the open end and the closed end, wherein the open end of each well is surrounded by the frame surface, and the closed end is between at least one wall and includes a well surface in contact with at least one wall, and at least one continuous ring on the well surface of the closed end of one or more wells. It is assumed that the continuous ring can be any shape, as long as the shape includes a continuous boundary at the closed end of the well. In some embodiments, the continuous ring is circular, elliptical, or other shape including a rounded boundary edge. In other embodiments, the continuous ring can be square, rectangular, triangular, or other geometric shape. In certain embodiments, at least one continuous ring 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 a continuous ring and is assumed to be any shape as long as it includes a continuous boundary. In some embodiments, a multiwell plate includes multiple continuous rings configured to define multiple cell seeding regions.

[0012] In another embodiment, the technology relates to a concentrator mask for seeding cells in a liquid medium into a multiwell plate. The concentrator mask includes a frame having a frame surface and frame sides extending from the frame surface, and a plurality of funnels extending from the frame surface, each funnel having a first open end and a second open end, the first open end being connected to the frame surface and having a larger diameter than the second open end.

[0013] Another aspect of the present technology relates to a method for seeding cells in the central portion of a culture well. This method includes pipetting a liquid medium containing cells into at least one cell seeding region surrounded by at least one continuous ring on the surface of the closed end of each well.

[0014] Another aspect of the present technology relates to a cell seeding system including a multiwell plate and a concentrator mask. The multiwell plate includes a frame having a frame surface and frame sides extending from the frame surface, and a plurality of wells, each well having an open end, a closed end opposite the open end, and at least one wall between the open end and the closed end, wherein the open end of each well is surrounded by the frame surface, and the closed end is between the walls and includes a well surface in contact with the walls, and at least one continuous ring on the well surface of the closed end of one or more wells. The concentrator mask includes a frame having a frame surface and frame sides 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, the first open end being connected to the frame surface and having a larger diameter than the second open end. The concentrator mask and the multiple funnels of the multiwell plate form a cell seeding system when the concentrator mask funnel is inserted into the multiple wells, such that the second open end of the funnel contacts a continuous ring on the well surface at the closed end of each well.

[0015] Another aspect of this technology relates to a method of seeding cells into the center of wells on a multiwell 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 in an area surrounded by at least one continuous ring on the well surface. [Brief explanation of the drawing]

[0016] [Figure 1A] This is a cross-sectional view showing a multi-well plate having wells containing a continuous ring on the inner bottom surface of the wells. [Figure 1B] Figure 1A is an enlarged view of the diagram, showing a multi-well plate with wells containing a continuous ring on the inner bottom surface of the wells. [Figure 2A] This is a schematic diagram of a concentrator mask for seeding cells into a multiwell plate. [Figure 2B] Figure 2A is a cross-sectional view showing a cross-section of a concentrator mask used for seeding cells into a multiwell plate. [Figure 3] This is a cross-sectional view of a cell seeding system including a concentrator mask inserted into a multiwell plate having wells containing a continuous ring on the inner bottom surface of the wells. [Figure 4] This is a schematic diagram of a 96-well plate having wells containing a continuous ring on the inner bottom surface of the wells. [Modes for carrying out the invention]

[0017] This instruction is best understood when read in conjunction with the attached drawings. Features are not necessarily depicted to a consistent scale. Wherever practical use is appropriate, similar reference numbers refer to similar features.

[0018] It should be understood that the technical terms used herein are intended solely to describe specific embodiments and are not intended to limit them. Defined terms are in addition to the technical and scientific meanings of defined terms that are generally understood and accepted in the art of this teaching.

[0019] 〔Definition〕 As used herein, the terms "substantial" or "substantially" mean, in addition to their ordinary meanings, within the tolerance or degree of those skilled in the art.

[0020] As used herein, the terms "substantially" and "about" mean within the tolerance or an amount corresponding to those skilled in the art. The term "about" generally refers to plus or minus 15% of the indicated numerical value. For example, "about 10" indicates a range of 8.5 to 11.5. For example, "substantially the same" means that those skilled in the art would consider the items being compared to be the same. In the present disclosure, numerical ranges include the numbers defining the range.

[0021] Before various embodiments are described, it should be understood that the teachings of the present disclosure are not limited to the specific embodiments described, and that the disclosure itself, of course, can vary. It should also be understood that the technical terms used herein are for the purpose of describing only specific embodiments and are not intended to limit, as the scope of the present teachings is limited only by the appended claims.

[0022] 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 belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present teachings, but some exemplary methods and materials are described herein. All patents and publications referred to herein are incorporated by reference expressly.

[0023] As used in the specification and the appended claims, the terms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise. Thus, for example, "a well" includes one well and a plurality of wells.

[0024] [Multi-well Plate with Wells Having Continuous Rings] In at least one embodiment, the state-of-the-art relates to a continuous ring formed at the bottom of each well of a cell culture or assay well plate. The continuous ring creates a central cell seeding region on the bottom of the well. When cells are seeded at the center of the ring, the ring functions as a physical barrier to confine the cells to the center of the well. This has several advantages outlined below.

[0025] Furthermore, when combined with certain micro-well assay devices such as the Agilent XF instrument, the continuous ring functions to define the volume of the assay micro-chamber, improving assay sensitivity. For example, in some embodiments, the continuous ring of the well joins with the analytical instrument to form a semi-sealed, temporary, reduced-volume micro-chamber in which metabolic measurements can be made.

[0026] In some embodiments of the present technology, cells are seeded at the center of the well within the boundary formed by the continuous ring. The continuous ring provides a physical barrier to confine the seeded cells to the center of the well. By seeding cells only at the center of the well, away from the sidewalls of the well, the effects caused by temperature differences and / or fluid differences near the perimeter of the well, as well as light transmission and cell seeding density gradients, are mitigated. Since the cells are at a distance from the sidewalls of the well, the effects due to differences in illumination are also minimized.

[0027] In other embodiments, the technique relates to a method for seeding cells inside a series of rings on the bottom of the wells of a multiwell plate. In some embodiments, cells in a liquid medium are added (e.g., by pipetting) to a cell seeding area defined by the series of rings. In these embodiments, the cells can then be grown, for example, by culture, but cell growth is limited to the cell seeding area formed by the series of rings. The multiwell plate and the method of using it described herein are compatible with both adherent and suspended cells. In some other embodiments, cells, tissues, or organoids can be added (e.g., seeded) to specific areas formed by the series of rings.

[0028] In other embodiments, cells can be seeded both inside and outside the continuous ring, for example, to create a non-contact co-culture of different cell types. In some embodiments, the cell seeding region includes a cell culture medium restricted to the cell seeding region by a continuous ring(s). In other embodiments, the cell seeding region includes a cell culture medium that is in fluid communication with cell culture media from other cell seeding regions.

[0029] In some embodiments, the wells of the multiwell plate of the present technology may be coated with a cell adhesion-promoting substance to facilitate the adhesion of suspended cells to the well surface or to improve the adhesion of adherent cells. For example, a polycationic coating, such as poly-1-lysine, may be used to promote the adhesion of any cell type to the well surface. In other embodiments, an uncoated multiwell plate may be used.

[0030] In additional embodiments, the technology relates to the preparation and maintenance of non-contact cell co-cultures of different cell types either inside or outside a continuous ring. The continuous ring for providing non-contact co-cultures may also be of any shape, as long as its shape includes a continuous boundary over the closed ends of the wells. In some embodiments, the continuous ring for non-contact co-cultures is circular, elliptical, or other shapes including rounded boundary edges. In other embodiments, the continuous ring for non-contact co-cultures may be square, rectangular, triangular, or other geometric shapes.

[0031] In certain embodiments, at least one continuous ring of any shape is configured to define at least one cell seeding region on the well surface. In other embodiments, a number of sets of continuous rings, such as but not limited to concentric rings, are configured to define multiple cell seeding regions on the well surface. In other embodiments, a physical barrier formed on the well surface may not be a ring, but instead be configured to provide a grid or other configuration of cell seeding regions.

[0032] If multiple cell seeding regions exist on the well surface, each cell seeding region may contain different cell types, the same cell type, a mixture of cell types, or any combination of the above as desired by those skilled in the art. In any case, the shape and number of cell seeding regions are defined by the shape and number of continuous rings and may be of any shape, size, and / or configuration in order to provide continuous boundaries and / or borders to the cell seeding regions and / or regions.

[0033] An additional embodiment of this technology relates to the use of microwell plates in combination with fiber optic probes, such as those used in Agilent XF instruments. In these embodiments, differences in optical signals caused by radially different optical signal transmission are minimized by seeding cells only in the center of the wells. This improves measurement sensitivity and results in a more uniform representation of signal transduction across the wells.

[0034] Without being constrained by theory, cells confined to the center of the well are thought to be less affected by temperature gradients within the well that occur during cell culture workflows and assays. Furthermore, because the cells are confined to the center of the well, light transmission remains at the highest point, enabling improved detection sensitivity and reduced assay variability introduced by cells seeded around the well.

[0035] In other embodiments, wells containing a continuous ring form a smaller assay microchamber, enabling better assay sensitivity when used with a microwell plate reader such as an Agilent XF instrument or other well-based measurement assays that have a radial dependence on signaling or on the assay cell seeding region or assay volume. Without being constrained by theory, it is also conceivable that cells seeded in the cell seeding region formed by the continuous ring exhibit reduced edge effects, allowing all wells in the well plate to be used in assays with improved interwell analysis and uniformity.

[0036] Figure 1A is a cross-sectional view showing a cross-section of an embodiment of the multiwell plate 101 having wells with continuous rings on the inner bottom surface of the wells. In this embodiment, the multiwell plate 101 is defined by a frame having a frame surface 102, frame sides 103, and a frame base 104. The frame in this embodiment also includes a frame tab 105 for multiwell plate processing. The multiwell plate 101 also includes well walls 106, open ends 107, and closed ends 108. The closed ends 108 of the wells further include a continuous ring 109 defining a cell seeding area 110. In this embodiment, the multiwell plate 101 also includes a perimeter well plate moat 111, whereas in other embodiments, the multiwell plate of the art does not include a perimeter well plate moat. Figure 1B is an enlarged view of Figure 1A and shows an embodiment of the multiwell plate having wells with continuous rings on the inner bottom surface of the wells.

[0037] In some embodiments, the cell seeding area surrounded by the continuous rings covers approximately 10% to 80% of the total well surface area. In other embodiments, the cell seeding area surrounded by the continuous rings covers approximately 55% to 75% of the total well surface area. In some embodiments, the cell seeding area surrounded by the continuous rings covers approximately 60% to 70% of the total well surface area. In other embodiments, the cell seeding area is approximately 65% ​​of the total well surface area. In other embodiments, the cell seeding area is approximately 10% to 25% of the total well surface area.

[0038] 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.

[0039] 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.

[0040] The multiwell plates of this technology can be configured in any form or orientation, including having dimensions that match the number and spacing of wells of a standard multiwell plate. For example, in some embodiments, the multiwell plates of this technology include at least 8 wells. In other embodiments, the multiwell plates of this technology include at least 24 wells. In some embodiments, the multiwell plates of this technology include at least 48 wells. In other embodiments, the multiwell plates of this technology include at least 96 wells. In other embodiments, the multiwell plates of this technology include at least 384 wells. In other embodiments, the multiwell plates of this technology include at least 1536 wells.

[0041] [Concentrator mask for cell seeding] In another embodiment, the technology provides a cell seeding and concentrating mask comprising a plurality of funnels. In some embodiments, the plurality of funnels are configured to allow funnel-shaped well inserts to be inserted into strips or grids of wells in a well plate. In some embodiments, the cell seeding and concentrating mask is used to restrict cell seeding to areas within wells smaller than the bottom of the wells. 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, the first open end being connected to the frame and having a larger diameter than the second open end of the funnel.

[0042] The cell seeding and concentrating mask of this technology allows for the seeding of cells into a selected area within a larger well using standard pipettes and techniques. In some embodiments, the cell seeding and concentrating mask restricts the seeded cells to the central well region during culture. The concentrating mask of this technology can be used to seed adherent cells or suspension cells.

[0043] In some embodiments, the wells of a multiwell plate may be coated with a cell adhesion-promoting substance to facilitate the adhesion of suspended cells to the well surface or to improve the adhesion of adherent cells. For example, a polycationic coating, such as poly-1-lysine, may be used to promote the adhesion of any cell type to the well surface. In other embodiments, an uncoated multiwell plate may be used.

[0044] In some embodiments, the second open end of the funnel is configured to be smaller than the bottom of the well into which the funnel is inserted. In these embodiments, the concentrator mask can be used to seed cells in the center of the well rather than at the well edge near the surrounding wall.

[0045] In certain embodiments, the technique relates to a method for pipetting cells in a liquid solution into the first open end of a concentrator mask funnel inserted into the wells of a multiwell plate, allowing the cells to settle. In other embodiments, the multiwell plate is rotated in a centrifuge so that the cells rotate onto an adhesive coating at the bottom of the wells. The multiwell plate is then transferred to an incubator to promote cell growth and proliferation. In some embodiments, the concentrator mask remains in the well plate during culture. In other embodiments, the concentrator mask may be removed from the well plate before culture. In additional embodiments, the concentrator mask remains in the well plate during culture and is removed before analysis of the seeded cells.

[0046] In another embodiment of this technology, the distal end of the second open end of the funnel creates an interface with the bottom of the well. In some embodiments, the interface is a liquid-tight seal, while in other embodiments, the interface allows or reduces the passage or diffusion of liquid by providing a gap, etc. In this embodiment, the cell solution is pipetted into the first open end of the funnel, and the solution fills the well both inside and outside the second open end of the funnel. Without being constrained by theory, it is assumed that cells settle to the bottom of the well by gravity, and therefore the number of cells deposited in the region at the bottom of the well depends on the number of cells suspended above it. Thus, the cell concentrator mask of this embodiment is designed so that cells physically occupy the volume above the region at the bottom of the well where they are undesirable. Therefore, the final result of cell seeding according to this embodiment of the technology is cells seeded at a high concentration in the center of the well, but cells are not seeded or are seeded at a low concentration in the peripheral region of the well near the well wall.

[0047] In certain embodiments, the outer diameter of the second open end of the funnel is configured to substantially match the inner diameter of the well into which the funnel is inserted, so 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 well or a portion thereof. The funnel of this technology can be configured to be inserted into wells of any size. Furthermore, in this embodiment, the size of the area into which high-concentration cells are seeded is determined by the inner diameter of the second open end of the funnel and the way in which the funnel is configured to be inserted into the wells of a multiwell plate.

[0048] In additional embodiments, the technology provides a cell concentrator mask comprising a funnel having a second open end, the second open end comprising a distal elastomer portion. In some embodiments, the distal elastomer portion of the second open end creates an interface with the bottom of the wells, which is a liquid-resistant seal. In this embodiment, the cell suspension can be pipetted into the first open end of the funnel and the bottom of the wells, and air can be replaced with liquid cell solution from the bottom of the funnel. In this embodiment, the cell suspension seeds cells only at the bottom of the wells inside the inner diameter of the second open end of the funnel. In this embodiment, the seeded cells settle at the bottom of the wells with the funnel inserted into the wells. The multiwell plate is then transferred to an incubator to promote cell growth and proliferation. In some embodiments, the concentrator mask remains on the well plate during culture. In other embodiments, the concentrator mask may be removed from the well plate before culture. In additional embodiments, the concentrator mask remains on the well plate during culture and is removed before analysis of the seeded cells. In certain embodiments of this technology, a region of enriched cells is seeded in the center of the well, while cells are not present around the well near the edges of the well wall.

[0049] In some embodiments, the distal elastomer portion of the second open end includes a compliant sealing material, such as an elastic, essentially fluid-impermeable material in the form of an O-ring. The compliant sealing material can be in any shape suitable for the end of the second open end. For example, the compliant sealing material may be a toroidal O-ring, a gasket with a rectangular cross-section, a metal gasket, or another type of compliant material. In one embodiment, the compliant sealing material may be a fluoroelastomer material or other material that forms a fluid seal with the opposing well surface. In another embodiment, the compliant sealing material is silicone rubber. In some embodiments, the compliant sealing material creates a radial seal between the second open end and the well surface. Other sealing orientations may also be considered. The compliant sealing material can be a variety of rubbers, e.g., fluoropolymers, bunan, EPDM, or possibly metallic with compliant overplating, depending on the temperature used and the components and conditions of the other cell culture medium. The compliant sealing material may also be coated with a chemically inert and biocompatible coating, if the O-ring material allows it.

[0050] In some embodiments, the concentrator mask of the present technology is configured to connect to flat-bottom cell culture wells. In other embodiments, the concentrator mask of the present technology is configured to connect to dimpled wells, such as Agilent XF wells.

[0051] In yet another embodiment, the concentrator mask of the technology is configured to bond to the well of the technology, which includes a continuous ring molded at the bottom of the well.

[0052] Therefore, the concentrator mask of this technology can be used in a method of seeding cells into a central cell seeding area in a larger well. In some embodiments, the concentrator mask funnel is used in a method of concentrating the cell seeding to the center of the bottom of the well. In some embodiments, the concentrator mask seedes the majority of cells above the area at the bottom of the central well at a desired cell concentration. In other embodiments, the concentrator mask of this technology can be used in a method of removing cell seeding from the area at the bottom of the well where cells are undesirable.

[0053] In some embodiments, the cell seeding and concentration mask of this technology can be used with a cell suspension in combination with centrifugation and surface coating so that the cells in the suspension adhere to the well surface. In certain embodiments, the concentrator mask can be removed before downstream analysis.

[0054] This method may further include cell analysis, such as analysis using the Agilent XF assay.

[0055] In some methods of this technology, the method further includes removing air trapped at the interface at the bottom of the concentrator mask / well by means of pipetting or the like.

[0056] In some embodiments, about 5.0 μl to about 20 μl of medium can be pipetteed onto the concentrator mask. In other embodiments, about 10 μl to about 15 μl of medium can be pipetteed onto the concentrator mask. In some embodiments, about 12.5 μl of medium can be pipetteed onto the concentrator mask. Thus, the methods of the latest technology can be performed using volumes that do not lead to significant pipetting errors.

[0057] In some embodiments of this technology, the seeded multiwell plate may be transferred to an incubator for cell proliferation. In some of these embodiments, the concentrator mask can remain in place during cell culture. Thus, certain embodiments of this technology are suitable for low-adhesion cells. Other embodiments of this technology relate to the culture of high-adhesion cells.

[0058] In other embodiments, the concentrator mask and the method of using it are compatible with commercially available multiwell plates such as Agilent XF well plates. In additional embodiments, the concentrator mask and the method of using it are compatible with state-of-the-art multiwell plates that include continuous rings on the inner bottom well surface.

[0059] Figure 2A is a schematic diagram of a concentrator mask for seeding cells into a multiwell plate. In this embodiment, the concentrator mask 201 is defined by a frame surface 202 and a frame side surface 203. In this embodiment, the frame also includes frame tabs 204 for aligning and stacking the concentrator mask onto the multiwell plate. Multiple funnels 205 extend from the frame. The funnels include a first open end 206 and a second open end 207, the first open end 206 having a larger diameter than the second open end 207. In some embodiments, the distal end 208 of the funnel includes an elastomer portion. In some embodiments, the funnel 205 is inserted into a well, creating a liquid-resistant interface between the bottom of the well and the distal end 208 of the funnel. Figure 2B is a schematic cross-sectional view of Figure 2A, showing a cross-section of the concentrator mask for seeding cells into a multiwell plate.

[0060] The concentrator mask of this technology can be configured in any form or orientation, including being compatible with a standardized multiwell plate and having an appropriate number and spacing of funnels to fit therein. For example, in some embodiments, the concentrator mask of this technology includes at least 8 funnels. In other embodiments, the concentrator mask of this technology includes at least 12 funnels. In other embodiments, the concentrator mask of this technology includes at least 24 funnels. In some embodiments, the concentrator mask of this technology includes at least 48 funnels. In other embodiments, the concentrator mask of this technology includes at least 96 funnels. In other embodiments, the concentrator mask of this technology includes at least 384 funnels. In other embodiments, the concentrator mask of this technology includes at least 1536 funnels.

[0061] In some embodiments, the concentrator mask of the present technology 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 technology 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 technology 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 technology is configured to include a funnel having an inner diameter of about 2.0 mm.

[0062] [Cell seeding system including multiwell plates and concentrator masks] This technology also relates to a cell seeding system, including a concentrator mask as described herein, used in combination with a continuous ring multiwell plate as described herein. In this embodiment, the multiple funnels of the concentrator mask are configured to fit into the multiple wells of the multiwell plate.

[0063] The cell seeding system of this technology 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 between the open end and the closed end, wherein the open end of each well is surrounded by the frame surface and the closed end is between the walls and includes a well surface in contact with the wall, and at least one continuous ring on the well surface of the closed end of each well.

[0064] The cell seeding system also includes a concentrator mask comprising a frame having a frame surface and frame sides extending from the frame surface, and a plurality of funnels extending from the frame surface, each funnel having a first open end and a second open end, the first open end being connected to the frame surface and having a larger diameter than the second open end.

[0065] In the cell seeding system of this technology, the multiple funnels of the concentrator mask are configured to fit multiple wells of a multiwell plate such that the second open end of the funnel contacts a continuous ring on the well surface at the closed end of each well. In some embodiments, the bottom portion 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.

[0066] Figure 3 is a schematic cross-sectional view of a cell seeding system, including a concentrator mask inserted into a multiwell plate having wells with continuous rings on the well surface at the closed ends of the wells. In this embodiment, the cell seeding system 301 is formed by a concentrator mask 201 and a multiwell plate 101 configured to fit together so that the funnel of the concentrator mask 201 is inserted into the wells of the multiwell plate 101. The cell seeding system 301 comprises a multiwell frame surface 102, a multiwell frame side surface 103, and a multiwell frame base 104. In this embodiment, the multiwell frame also includes multiwell frame tabs 105 for plate processing. The multiwell plate comprises well walls 106 (invisible) defining the wells and, in this embodiment, a perimeter well plate moat 111. The multiwell plate wells also include an open end (invisible) and a closed end 108, which include a continuous ring 109. The continuous ring 109 forms a cell seeding region 110 on the central portion of the closed well end 108.

[0067] The concentrator mask 201 of the cell seeding system 301 comprises a frame surface 202 and frame sides 203. The concentrator mask also includes a plurality of funnels 205. The funnels include 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 the wells of the multiwell plate 101. In this embodiment, the second open end 207 of the funnel is configured to contact a continuous ring 109 on the well surface of the closed end 108 of the well. 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 creates a liquid-tight seal. In certain 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 multiwell plate frame tab 105 for plate processing. The cell seeding system also includes a concentrator mask frame tab 204 for aligning and inserting the concentrator mask 201 into a multiwell plate 101.

[0068] The technology also relates to a method of using a cell seeding system 301 to seed cells in the center of the wells of a multiwell plate. In some embodiments, the method involves adding a liquid medium containing cells to the first open funnel end 206 of the cell seeding system, so that the cells are deposited in a cell seeding region 110 defined by at least one continuous ring 109 on the well surface at the closed end 108 of the well. In some embodiments, the seeded cell seeding system 301 may be transferred to an incubator for cell proliferation. In certain embodiments, the concentrator mask 201 may be removed from the multiwell plate 101. In other embodiments, the concentrator mask 201 may remain 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 before culturing. In other embodiments, the concentrator mask 201 may remain inserted in the multiwell plate 101 while the cells are being cultured for proliferation. In some embodiments, the concentrator mask 201 can be removed from the multiwell plate 101 before cell analysis. In other embodiments, the concentrator mask 201 can remain on the multiwell plate 101 during cell analysis. In some embodiments, cell analysis includes optical reading.

[0069] This technology relates to and is compatible with 6, 12, 24, 48, 96, 384, and 1536 well formats. Figure 4 is a schematic diagram of a 96-well plate of this technology, having wells with continuous rings on the inner bottom surface of the wells. In this embodiment, the multiwell plate 401 is defined by a frame having a frame surface 102, frame sides 103, and frame base 104. The multiwell plate 401 also includes well walls 106, open ends 107, and closed ends 108 (not visible). The closed ends 108 of the wells further include continuous rings 109 defining a cell seeding area 110. In this embodiment, the multiwell plate 401 does not include a perimeter well plate moat or frame tab for plate processing, but in other embodiments, the multiwell plate of this technology includes a perimeter well plate moat and frame tab for processing.

[0070] [Metabolic measurement] In some embodiments, the methods, apparatus, and systems are useful for measuring cell biology, particularly in the area of ​​micro-respiratory measurements, which include quantitatively measuring the bioenergy or metabolic state of a small number of cells, as opposed to respiratory measurements performed on the whole animal. Historically, micro-respiratory measurements were performed using milliliter cell cultures and microscope glass flow cells with Clark electrodes to measure cellular metabolism. This approach is not microscopic, easy, or high-throughput. Seahorse Bioscience's flux analyzers and assays have improved the technique of micro-respiratory measurements by introducing comprehensive assays that can be easily performed on 8, 24, and 96 plastic cell culture plates. The resulting techniques enable the complex characterization of both the glycolysis and oxidative phosphorylation pathways by introducing various stimulants, inhibitors, and custom drugs and measuring changes in oxygen consumption and proton production. Additional details regarding micro-respiratory measurements are provided in U.S. Patent Application No. 15 / 896,255, which is incorporated herein by reference in its entirety.

[0071] In another aspect of the present invention, the method, apparatus, and system are used for measuring the metabolism of individual cell types in culture. In some embodiments, the method, apparatus, and system can be used for the analysis of 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 inside a cell seeding area surrounded by a continuous ring, and a second cell type between the continuous ring and the well wall, outside the continuous ring. In some embodiments, different cell types in non-contact co-culture are in fluid communication with each other. In other embodiments, different cell types in non-contact co-culture are not in fluid communication with each other.

[0072] An additional aspect of the present invention includes a plurality of continuous rings of any shape, which provide a number of cell seeding regions or segments of any size, shape or configuration for the preparation and maintenance of non-contact co-cultures by seeding different cells into different cell seeding regions / segments. Furthermore, certain aspects of the technology include molded physical barriers on the well surface, which are not rings but are configured to provide a grid or alternative configuration of cell seeding regions. When the non-contact co-culture of the technology is produced by seeding different cell types into different cell seeding regions, the method, apparatus, and system can be used to obtain metabolic measurements independently from one cell seeding region / segment at a given time. Alternatively, the apparatus and system of the method 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.

[0073] The present method, apparatus, and system can measure the physiological properties of a cell population using one or more sensors. The sensors may be fluorescence sensors, emission sensors, ISFET sensors, surface plasmon resonance sensors, sensors based on the optical diffraction principle, sensors based on Wood's anomalous principle, acoustic sensors, or microwave sensors. The technique is not limited to specific cell assays, measurements, or sensors, but can instead be used by those skilled in the art in combination with any desired cell analysis approach. Accordingly, the present system, apparatus, and method may include one or more of the aforementioned sensors arranged to measure one or more properties of a sample in a well described herein.

[0074] This method, apparatus, and system can be used in various 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, this device can be used to screen various molecules for their effects on cellular metabolism in co-culture, protein secretion, or intracellular / extracellular ion exchange. This method, apparatus, and system can also be used to determine the health of cells in culture, including co-culture, both before and after conventional assays are performed, thereby improving the performance of such assays.

[0075] [Cell population] The cell population used in this method and apparatus may include any cells of interest. Such 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 blood vessels. At least a portion of the cells may be suspended in a culture medium. At least a portion of the cells may include biological tissue, organoids, spheroids, or cultured tissue. In some embodiments, at least a portion of the cells are attached to the closed end or wall of the well.

[0076] Known cell lines can be used as cell types in this method, apparatus, and system. For example, known cell lines that can be used in combination with this technology include C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa, HeLa-S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Pancl, 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 epithelium, BALB / 3T3 mouse embryonic fibroblasts, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B3 5, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR293, BxPC3, C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO- K1, CHO-K2, CHO-T, CHODhfr- / -, 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, Hepalclc7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYOl, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-1OA, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK This includes, but is not limited to, II, MOR / 0.2R, MONO-MAC6, 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 / PNT2, 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 various sources known to those skilled in the art (see, for example, the American Type Culture Collection (ATCC) (Manassas, Virginia)). These or other cell lines can be used as the first or second cell type in this method and apparatus. In some embodiments, the first cell type is a cell population taken from a subject (such as a human patient), and the second cell type is a known cell line.

[0077] [Exemplary Embodiments] 1. A multi-well plate for cell populations in liquid culture medium, A frame having a frame surface and frame sides extending from the frame surface, A plurality of wells, each having an open end, a closed end opposite the open end, and at least one wall between the open end and the closed end, wherein the open end of each well is surrounded by a frame surface, and the closed end is between at least one wall and includes a well surface in contact with at least one wall, A multiwell plate comprising at least one continuous ring on the well surface at the closed end of one or more wells. 2. A multiwell plate of Embodiment 1, wherein at least one continuous ring is configured to define at least one cell seeding region on the well surface, which can work in cooperation with a lid, plunger, or other element to form an assay microchamber. 3. A multiwell plate according to Embodiment 1, wherein the multiwell plate includes a plurality of continuous rings configured to define a plurality of cell seeding regions. 4. A multiwell plate according to any of the above embodiments, wherein at least one continuous ring has a height of about 0.01 mm to about 2 mm. 5. A multiwell plate according to any of the above embodiments, wherein at least one continuous ring has an inner diameter of about 0.5 mm to about 6.0 mm, for example, an inner diameter of about 2.0 mm. 6. A concentrator mask for seeding cells in liquid culture medium into a multiwell plate, A frame having a frame surface and frame sides extending from the frame surface, A plurality of funnels extending from the surface of a frame, each funnel having a first open end and a second open end, including A concentrator mask having a first open end connected to the frame surface and having a larger diameter than a second open end. 7. A concentrator mask of Embodiment 6, wherein the second open end includes a distal elastomer portion. 8. A concentrator mask of embodiment 6 or 7, wherein the concentrator mask includes at least eight funnels. 9. Concentrator masks of embodiments 6 to 8, wherein the funnel has an inner diameter of approximately 0.5 mm to approximately 6.0 mm, for example, an inner diameter of approximately 2.0 mm. 10. A method for seeding cells in the central part of a culture well, A method comprising adding a liquid culture medium containing cells to at least one cell seeding area on the well surface of any of Embodiments 1 to 5. 11. The method of Embodiment 10, wherein the culture wells are transferred to an incubator, and the cells are seeded and then cultured. 12. The method of Embodiment 11, wherein the cultured cells substantially do not exhibit or show any edge effects induced by the incubator. 13. A cell seeding system comprising a multiwell plate and a concentrator mask, The multiwell 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 between the open end and the closed end, wherein the open end of each well is surrounded by the frame surface, and the closed end is between the walls and includes a well surface in contact with the wall, and at least one continuous ring on the well surface of the closed end of each well, The concentrator mask comprises a frame having a frame surface and frame sides extending from the frame surface, and a plurality of funnels extending from the frame surface, each funnel having a first open end and a second open end, the first open end being connected to the frame surface and having a larger diameter than the second open end. A cell seeding system in which multiple funnels are configured to fit multiple wells, and as a result, the second open end of the funnels forms an interface with a continuous ring on the well surface of the closed end of one or more wells. 14. The system of Embodiment 13, wherein the interface between the second open end and the continuous ring is a gap small enough to reduce the diffusion of the liquid. 15. The system of Embodiment 13, wherein the second open end includes a distal elastomer portion. 16. The system of Embodiment 15, wherein the interface is physical contact between the distal elastomer portion of the second open end and the continuous ring, thereby forming a liquid-tight seal. 17. A method for seeding cells into a multiwell plate, A method comprising adding a liquid culture medium containing cells to the first open end of a cell seeding system of Embodiment 13, wherein the cells are deposited in an area surrounded by at least one continuous ring on the well surface. 18. The method of Embodiment 17, wherein after seeding the cells, the multiwell plate is transferred to an incubator. The cultured cells are considered to exhibit no or substantially no edge effect induced by the incubator. 19. The method of Embodiment 18, wherein the concentrator mask is removed from the multiwell plate before placing the multiwell plate in the incubator. 20. The method of Embodiment 17, further comprising analyzing cells in a multiwell plate to obtain optical measurements.

[0078] [Example 1] Cells were seeded, grown, and analyzed in multi-well plates containing continuous rings using this technology. These cells were compared to control cells seeded, grown, and analyzed in multi-well plates containing standard wells without continuous rings. The wall height of the tested continuous rings was 0.2 mm and the inner diameter was 2.0 mm. The same number of cells (4,500) were analyzed in both well types.

[0079] After seeding and growing the cells, the oxygen consumption rate (OCR) was measured using an Agilent XFp instrument. The rate was measured by obtaining optical measurements. The results showed that the same number of cells (4,500) produced a three-fold higher signal intensity when cultured in wells containing a continuous ring compared to cells cultured in standard wells without a continuous ring.

[0080] In consideration of this disclosure, it should be noted that the methods and apparatus can be carried out in accordance with this teaching. Furthermore, various components, materials, structures, and parameters are included only as examples and illustratively, and not in an restrictive sense. In consideration of this disclosure, this teaching can be carried out for other uses, and the components, materials, structures, and equipment for carrying out these uses can be determined while remaining within the scope of the appended claims.

Claims

1. A concentrator mask for seeding cells in a liquid culture medium into a multiwell plate, A frame having a frame surface and frame sides extending from the frame surface, At least one funnel extending from the frame surface, each of the at least one funnel including a first open end connected to the frame surface and a second open end having a smaller diameter than the first open end, and each of the at least one funnel including a straight tubular portion extending linearly from a spaced portion spaced apart from both the first and second open ends to the first open end, and an expanding portion extending from the spaced portion to the second open end, which gradually widens in diameter from the second open end toward the spaced portion, Concentrator mask, including

2. The concentrator mask according to claim 1, wherein the distal end of at least one funnel includes an elastomer portion.

3. The concentrator mask according to claim 1, wherein the concentrator mask includes at least eight funnels.

4. The concentrator mask according to claim 1, wherein the funnel has an inner diameter of about 0.5 mm to about 6.0 mm.

5. A cell seeding system comprising a multiwell plate and a concentrator mask, The multiwell plate includes a frame having a frame surface and a frame side extending from the frame surface; a plurality of wells, each having an open end, a closed end opposite the open end, and at least one wall between the open end and the closed end, wherein the open end of each well is surrounded by the frame surface, and the closed end is between the walls and includes a well surface in contact with the walls; and at least one continuous ring on the well surface of the closed end of each well. The concentrator mask includes a frame having a frame surface and frame sides extending from the frame surface, and at least one funnel extending from the frame surface, each of the at least one funnel including a first open end connected to the frame surface and a second open end having a smaller diameter than the first open end, and each of the at least one funnel includes a straight tubular portion extending linearly from a spaced portion spaced apart from both the first and second open ends to the first open end, and an enlarged portion extending from the spaced portion to the second open end, which gradually widens in diameter from the second open end toward the spaced portion, A cell seeding system in which the at least one funnel is configured to fit into at least one of the plurality of wells, such that the second open end of the funnel forms an interface with the continuous ring on the well surface of the closed end of one or more of the wells.

6. The system according to claim 5, wherein the interface between the second open end and the continuous ring is a gap sufficient to reduce the diffusion of the liquid.

7. The system according to claim 5, wherein the distal end of at least one funnel includes an elastomer portion.

8. The system according to claim 7, wherein the interface between the elastomer portion of the second open end and the continuous ring forms a liquid-tight seal.

9. A method for seeding cells into a multiwell plate, A method comprising adding a liquid culture medium containing cells to the first open end of the cell seeding system according to claim 5, wherein the cells are deposited in a region on the well surface surrounded by the at least one continuous ring.

10. The method according to claim 9, wherein after seeding cells, the multiwell plate is transferred to an incubator.

11. The method according to claim 10, wherein the concentrator mask is removed from the multiwell plate before transferring the multiwell plate to the incubator.

12. The method according to claim 9, further comprising analyzing the cells in the multiwell plate to obtain optical measurements.

Images