Method and apparatus for cell culture well plates

Abstract

A multi-well plate is provided that takes into account some of the challenges that arise from seeding cells across the entire bottom surface of the well and addresses the physical and biological challenges associated with cells growing near the sidewalls of the well and the optical detection of such cells. [Solution] A multiwell plate for cell populations in liquid culture, comprising: a frame having a frame surface 102 and frame sides 103 extending from the frame surface; a plurality of wells, each well having an open end 107, a closed end 108 opposite the open end, and at least one wall 106 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 at least one wall and includes a well surface in contact with the at least one wall; and at least one continuous ring 109 on the well surface of the closed end of one or more wells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 928,12, filed October 30, 2019. No. 6,239,999, filed on Oct. 1, 2003, the contents of which are incorporated herein by reference.

[0002] The present invention relates generally to methods, devices, and systems for cells in culture. Specifically, the present invention relates to novel multi-well plates and concentrator masks. Ming also developed multi-well plates and concentrator mass spectrometers for cell seeding and cell assays. Regarding the use of [Background technology]

[0003] Multiwell plates are widely used for parallel and / or simultaneous cell measurements. Used by Agilent Technologies, Sigma-Aldrich, Various well formats from vendors such as ch, Thomas Scientific Multiwell plates for tissue culture are commercially available in 6, 12, 24, 48, and 96 wells. Coated plates available in 1536-, 384-, and 1536-well formats Coated and uncoated plates are available for adherent and suspension cell culture, respectively. .

[0004] Multiwell plates are often used to perform cell population measurements. When seeding cells into wells of a cell culture plate, the cell solution is pipetted into the wells. The cells settle and cover the bottom of the wells. The seeded well plate is then used for cell growth and proliferation. They are often placed in an incubator to promote proliferation. However, there are some drawbacks that result from seeding cells on the entire bottom of the well. There are several challenges, many of which are related to the spatial sensitivity associated with many in vitro cell assays. It depends on the degree.

[0005] Therefore, the cultured cells should be concentrated in the center of the well to improve optical assay sensitivity and edge induction. This includes cell seeding bias dyes to counter thermal heterogeneity associated with induced cell biological effects. It is often desirable to avoid spatial biases, including but not limited to:

[0006] For assays with optical readout, the efficiency of light transmission from the peripheral area of ​​the well is typically This is due to the reduced optical access near the sidewalls of the well. Furthermore, illumination and / or data collection efficiency may vary depending on the detector field of view. It is blocked by a ledge.

[0007] In some cases, plate reader strategies are used to generate uniform illumination across the wells. However, such a strategy is time-consuming and inefficient. Other plate reader strategies involve placing the plate in the center of the well to avoid areas of poor light transmission. This approach allows for measurements only from cells growing outside the center of the well. Relevant data may be omitted.

[0008] Furthermore, when cells are seeded in a well plate, the cells in the wells on the periphery of the well plate Cells in the wells around the perimeter of the plate may grow and behave differently than cells in the non-perimeter wells. This phenomenon is commonly called the "edge effect." Because this edge effect is important, It is common practice not to seed cells into the peripheral wells of multi-well plates. Without restraint, edge effects occur when the plate is placed in the incubator. This is thought to be due to differential heating of the well medium within the wells around the perimeter of the plate. Cells tend to gather on the side walls of the wells in response to temperature gradients. In the case of wells, the temperature gradient is more severe, leading to uneven cell growth towards the side walls of the well. When cells exhibiting edge effects are analyzed using optical assay techniques, well-to-well variability is observed. Some well plates have a hole in them to reduce the edge effect. It contains a mote that the user fills with medium, which provides heat and humidity to the surrounding well. It is thought to act as a buffer against

[0009] Cells growing near the sidewalls of a well and the physics associated with optical detection of such cells A device for culturing cells in multi-well plates that addresses environmental and biological challenges There remains a strong need in the art for methods and systems. Summary of the Invention

[0010] These and other features and advantages of the method and apparatus are set forth in the appended claims. As will become apparent from the detailed description below.

[0011] In one aspect, the present technology relates to a multi-well plate for cell populations in liquid medium. The multi-well plate is made of a frame surface and frame sides extending from the frame surface. a frame having a well, each well having an open end, a closed end opposite the open end, and a frame having a well having a closed end and a well having an open end and a closed end; a plurality of wells having at least one wall therebetween, the open end of each well being The closed end is surrounded by a frame surface and is between at least one wall and at least Multiple wells, including a well surface contacting one wall, and closure of one or more wells and at least one continuous ring on the end well surface. It is envisioned that the continuous rings can be of any shape, as long as they contain a continuous boundary. In an embodiment, the continuous ring may be circular, oval, or have other shapes that include rounded boundary edges. In other embodiments, the continuous rings are square, rectangular, triangular, or other geometric shapes. In certain embodiments, at least one continuous ring is formed on the well surface. The shape of the cell seeding region is configured to define at least one cell seeding region thereon. , is defined by the shape of a continuous ring and is assumed to be any shape as long as it contains a continuous boundary. In some embodiments, the multi-well plate defines multiple cell seeding regions. The device includes a plurality of continuous rings configured to

[0012] In another aspect, the present technology provides a method for seeding cells in liquid medium into multi-well plates. The concentrator mask has a frame surface and a frame extending from the frame surface. a frame having a rim side; and a plurality of funnels extending from a surface of the frame, each funnel having a a funnel having a first open end and a second open end, the first open end being connected to the frame; It is connected to the surface and has 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. The liquid medium containing the cells is distributed in at least one continuous ring on the surface of the closed end of each well. and pipetting the mixture into at least one cell seeding area surrounded by the mixture.

[0014] Another aspect of the present technology is a cell seeding system including a multi-well plate and a concentrator mask. The multi-well plate is made of a frame surface and a frame extending from the frame surface. a frame having sides, and each well having an open end, a closed end opposite the open end, and a a plurality of wells having at least one wall between an open end and a closed end, The ends are surrounded by the frame surface, and the closed end is a well between and in contact with the walls. a plurality of wells, including a surface, and at least one well surface at the closed end of the wells; The concentrator mask includes a frame surface and a continuous ring extending from the frame surface. a frame having a frame side 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 attached to the frame surface. The concentrator mask and the multi-wafer are connected to each other and have a larger diameter than the second open end. The multiple funnels in the plate are connected to the surface of the wells at the closed end of each well, with the second open end of the funnel. The cells are drawn when the funnel of the concentrator mask is inserted into multiple wells so that it comes into contact with the connecting ring. Form a seeding system.

[0015] Another aspect of the present technology is to introduce a liquid medium containing cells into a first open end of a funnel of a cell seeding system. Seed the cells in the center of the wells on the multi-well plate by pipetting. In the method, the cells are arranged in an area surrounded by at least one continuous ring on the surface of the well. It deposits in the area. [Brief explanation of the drawings]

[0016] [Figure 1A] FIG. 1 is a cross-sectional view showing a cross section of a multiwell plate having wells that include a continuous ring on the inside bottom surface of the well. [Figure 1B] 1A is an enlarged view of FIG. 1A showing a multiwell plate having wells containing a continuous ring on the inside bottom surface of the well. [Figure 2A] FIG. 1 is a schematic diagram of a concentrator mask for seeding cells into multi-well plates. [Figure 2B] FIG. 2B is a cross-sectional view of FIG. 2A showing a cross section of a concentrator mask for seeding cells into a multiwell plate. [Figure 3] FIG. 1 is a cross-sectional view of a cell seeding system including a concentrator mask inserted into a multiwell plate having wells that include a continuous ring on the inside bottom surface of the wells. [Figure 4] FIG. 1 is a schematic diagram of a 96-well plate having wells containing a continuous ring on the inside bottom surface of the well. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present teachings are best understood from the following detailed description when read in conjunction with the accompanying drawing figures. Features are not necessarily drawn to scale. Whenever practical, Like reference numbers refer to like features.

[0018] The terminology used herein is for the purpose of describing particular embodiments only. It should be understood that the definitions are not intended to be limiting. The technical and scientific definitions of terms generally understood and accepted in the art. It is an addition to the meaning.

[0019] [Definition] As used herein, the terms "substantial" or "substantially" mean In addition to the ordinary meaning, it means within the range or degree of tolerance of a person skilled in the art.

[0020] As used herein, the terms "approximately" and "about" mean within a reasonable range or The term "about" generally refers to a value that is plus or minus the stated numerical value. indicates minus 15%. For example, "about 10" indicates a range of 8.5 to 11.5. For example, "substantially the same" means that one of ordinary skill in the art would consider the items being compared to be the same. In this disclosure, numerical ranges are inclusive of the numbers defining the range.

[0021] Before various embodiments are described, it is understood that the teachings of the present disclosure are not limited to the particular embodiments described. It should be understood that the terms "specialty" and "value" are not intended to be limiting and as such may, of course, vary. The terminology used herein is for the purpose of describing particular embodiments only, and the scope of the present teachings is limited only by the accompanying It is understood that no limitation is intended, since it is limited only by the scope of the claims. sea ​​bream.

[0022] Unless otherwise defined, all technical and scientific terms used herein are defined by the It has the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present teachings. Although some exemplary methods and materials can be used for All patents and publications referenced herein are expressly incorporated by reference. There are.

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

[0024] Multiwell plates with wells having a continuous ring In at least one embodiment, the current technology involves the use of cell culture or assay well plates. The continuous ring is molded into the bottom of each well of the container. The continuous ring has a center Once the cells are seeded in the center of the ring, the ring will seed the cells into the well. It acts as a physical barrier to keep the object at the center of the hole. This involves several steps, outlined below. There are some advantages.

[0025] Additionally, it can be combined with certain microwell assay instruments, such as the Agilent XF instruments. When combined, the continuous ring functions to define the volume of the assay microchamber, Improved assay sensitivity. For example, in some embodiments, a continuous ring of wells A semi-sealed, temporary, reduced volume chamber that can be interfaced with analytical equipment to perform metabolic measurements. A small microchamber is formed.

[0026] In some embodiments of the present technology, the cells are grown within the boundary formed by the continuous ring. The continuous ring serves to constrain the seeded cells to the center of the well. By seeding cells only in the center of the well, away from the side walls of the well, This may affect light transmission and cell seeding density due to temperature and / or fluid differences around the periphery of the well. This reduces the effect caused by temperature gradients. This minimizes the impact of differences in lighting.

[0027] In other embodiments, the present technology involves the deposition of ions in a continuous ring on the bottom of a well of a multi-well plate. In some embodiments, the cells in the liquid medium are , is added (e.g., pipetted) to the cell seeding area defined by the continuous ring. In these embodiments, the cells can then be expanded, for example, in culture. However, cell proliferation is restricted to the cell seeding area formed by the continuous ring. The multiwell plate described, and the method of using it, is suitable for the culture of adherent and suspension cells. In some other embodiments, the cells, tissues, or organoids are compatible with both the The dye can be added (e.g., seeded) in a specific area formed by successive rings. do.

[0028] In other embodiments, the cells are cultured in a continuous manner, such as to create non-contact co-cultures of different cell types. The cells can be seeded both inside and outside the ring. The seeding area contains cell culture medium that is restricted to the cell seeding area by continuous ring(s). In other embodiments, the cell seeding regions are in fluid communication with cell culture medium from other cell seeding regions. Contains the cell culture medium being passed through.

[0029] In some embodiments, the wells of the multi-well plates of the present technology may include a surface cell adhesion, to facilitate the attachment of suspension cells, or to improve the adhesion of adherent cells For example, polycationic coatings, e.g. Poly-l-lysine is used to promote the attachment of any cell type to the surface of the well. In other embodiments, uncoated multiwell plates may be used. This can be done.

[0030] In additional embodiments, the present technology can be applied to different fine lines on either the inside or outside of a continuous ring. This invention relates to the preparation and maintenance of non-contact cell co-cultures of the cytoplasmic type. The ring may be of any shape, so long as that shape includes a continuous boundary on the closed end of the well. In some embodiments, the continuous ring for non-contact co-culture is circular, It may be oval or other shape with rounded border edges. The continuous ring for contact co-culture can be square, rectangular, triangular, or other geometric shapes. It is possible.

[0031] In certain embodiments, at least one continuous ring of any shape is present on the well surface. In other embodiments, the concentric rings are configured to define at least one cell seeding area. A number of sets of continuous rings, such as but not limited to rings, may be placed on the surface of a well. In another embodiment, the well surface is configured to define a cell seeding area. The physical barrier is not a ring; instead, a grid or other configuration of the cell seeding area is provided. The device may be configured to:

[0032] When there are multiple cell seeding areas on the well surface, each cell seeding area contains a different cell type. , may contain the same cell type, a mixture of cell types, or any combination of the above as desired by one of skill in the art. In any case, the shape and number of cell seeding regions depend on the shape and number of continuous rings. and provide a continuous boundary and / or border for the cell seeding area and / or region. It is contemplated that the device may be of any shape, size, and / or configuration.

[0033] Additional embodiments of the present technology may involve optical probes such as those used in Agilent XF instruments. This relates to the use of microwell plates in combination with fiber probes. In their embodiment, cells are seeded only in the center of the well, resulting in radially distinct optical signals. Minimize the optical signal difference caused by signal transmission, thereby improving measurement sensitivity. This results in more uniformly expressed signaling across the well.

[0034] Without being bound by theory, cells confined to the center of the well are essential for the cell culture workflow. It is also thought that the influence of temperature gradients within the wells that occur during the assay is minimal. Therefore, the cells are confined to the center of the well, where light transmission is highest and the cells remain in the well. Increased detection sensitivity and reduced assay variability introduced by cells seeded around the This becomes possible.

[0035] In other embodiments, the wells containing the continuous rings exhibit radial dependence or signal transduction. Agilent X with radial dependence on assay cell seeding area or assay volume Use a microwell plate reader such as an F instrument or other well-based measurement assay. to form smaller assay microchambers that allow for greater assay sensitivity Without being bound by theory, the cells seeded in the cell seeding area formed by the continuous ring The cells are then collected in all wells of the well plate, improving well-to-well analysis and uniformity. It is also believed that the fluorophores may exhibit reduced edge effects, allowing them to be used in controlled assays.

[0036] FIG. 1A shows the multiwell plate having wells containing a continuous ring on the inside bottom surface of the well. 1 is a cross-sectional view showing a cross section of an embodiment of plate 101. In this embodiment, The frame 101 includes a frame surface 102, a frame side 103, and a frame base 104. The frame of this embodiment is also defined by a multi-well plate. The multi-well plate 101 also includes a frame tab 105 for rate processing. The well includes a well wall 106, an open end 107, and a closed end 108. The closed end 108 of the well is It further includes a continuous ring 109 that defines a seeding area 110. In this embodiment, the multi- The well plate 101 also includes a surrounding well plate moat 111, although in other embodiments The multiwell plate of this technology does not include a peripheral well plate moat. 1B is an enlarged view of FIG. 1A, showing the present multi-well device having wells with continuous rings on the inside bottom surface of the wells. 1 shows an embodiment of a chiwell plate.

[0037] In some embodiments, the cell seeding area surrounded by the continuous ring is In another embodiment, the surface area is about 10% to about 80% of the total surface area. The cell seeding area surrounded by the well covers about 55% to about 75% of the total area of ​​the well surface. In some embodiments, the cell seeding area surrounded by the continuous ring comprises In another embodiment, the cell seeding area is about 60% to about 70% of the total surface area of ​​the well. In another embodiment, the cell seeding area is about 65% of the total area of ​​the well surface. It is about 10% to about 25% of the total area of ​​the well surface.

[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 is about 0.1 mm to about 0.5 mm or about 1 mm high. 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 another embodiment, 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 have the same number of wells and spacing as standard multiwell plates. The slits can be configured in any manner or orientation, including having dimensions that match the slits. For example, in some embodiments, the multiwell plates of the present technology contain at least eight In other embodiments, the multi-well plate of the present technology comprises at least 24 wells. In some embodiments, the multi-well plate of the present technology comprises at least In other embodiments, the multi-well plate of the present technology comprises at least 48 wells. In other embodiments, the multi-well plates of the present technology contain at least 96 wells. In other embodiments, the multi-well plate of the present technology comprises at least 384 wells. Contains at least 1536 wells.

[0041] [Cell seeding concentrator mask] In another aspect, the present technology provides a cell-seeding concentration mask that includes multiple funnels. In some embodiments, the plurality of funnels may be configured to connect funnel-shaped well inserts to the wells within the well plate. In some embodiments, the ion exchanger is configured to be inserted into a strip or grid of wells. The cell seeding concentrator mask restricts cell seeding to an area within the well that is smaller than the bottom of the well. In some embodiments, the concentrator mask comprises a frame and a frame. and a plurality of funnels extending from the mask. In another embodiment, the funnels of the concentrator mask include a first The first open end is connected to the frame, and the second open end is connected to the frame. 2 has a larger diameter than the open end of

[0042] The cell seeding concentration mask of this technology allows for larger volumes to be seeded using standard pipettes and techniques. In some embodiments, the cells are seeded in selected areas within the well. The cell seeding concentrator mask confines the seeded cells to the central well area during culture. The surgical concentrator mask can be used for seeding adherent or suspension cells.

[0043] In some embodiments, the wells of the multi-well plate are configured to allow suspension cells to be deposited on the well surface. To facilitate cell adhesion or to improve adhesion of adherent cells, For example, polycationic coatings, e.g., poly- L-lysine can be used to promote the attachment of any cell type to the surface of the well. In this embodiment, uncoated multiwell plates can be used. do.

[0044] In some embodiments, the second open ends of the plurality of funnels are at the bottom of the well into which the funnels are inserted. In these embodiments, the concentrator mask is configured to be smaller than the well. Used to seed cells in the center of the well, rather than near the edge of the well, near the perimeter wall. It is possible.

[0045] In certain embodiments, the present technology involves the step of culturing cells in a liquid solution in the wells of a multi-well plate. The cells are pipetted into the first open end of the concentrator mask funnel, which is inserted into the In another embodiment, the method comprises: Spin the multiwell plate in a centrifuge so that the plates rotate upward. The well plate is transferred to an incubator to promote cell growth and proliferation. In embodiments, the concentrator mask remains on the well plate during incubation. The concentrator mask may be removed from the well plate prior to incubation. The condenser mask remains on the well plate during culture and is removed prior to analysis of the seeded cells. .

[0046] In another embodiment of the present technology, the distal end of the second open end of the funnel forms an interface with the bottom of the well. In some embodiments, the interface is a liquid-tight seal, while in other embodiments, the interface is a The term "interface" refers to a surface that allows but reduces the passage or diffusion of liquid, such as by providing a gap. In this embodiment, the cell solution is pipetted into the first open end of the funnel, and the solution is , filling the well both inside and outside the second open end of the funnel. Instead, the cells settle to the bottom of the well by gravity and therefore settle in the bottom area of ​​the well. The number of cells that can be collected is thought to depend on the number of cells suspended above it. The cell concentrator mask of this embodiment separates the volume above the area of ​​the bottom of the well where cells are not desired. Therefore, seeding of cells according to this embodiment of the technique The end result is cells seeded at a high density in the center of the well, but cells near the well walls. The surrounding area is not seeded with cells or at a low concentration.

[0047] In certain embodiments, the outer diameter of the second open end of the funnel is equal to the inner diameter of the well into which the funnel is inserted. configured to substantially match, so that the inserted funnel is not subject to compression or interference fixation In some embodiments, the outer diameter of the well or part thereof is The funnel of the present technology can be configured to provide a predetermined gap with the inner diameter. Furthermore, in this embodiment, the cells can be inserted into a well. The size of the area where the cells are seeded is determined by the inner diameter of the second open end of the funnel and the size of the funnel when the funnel is multi-well. This is determined by the way in which it is configured to be inserted into the wells of the plate.

[0048] In an additional embodiment, the present technology provides a cell concentrator mask including a funnel having a second open end. and the second open end includes a distal elastomeric portion. The distal elastomeric portion of the open end of the creates an interface with the bottom of the well that is a liquid-tight seal. In this embodiment, the cell suspension is pipetted into the first open end of the funnel and into the bottom of the well. The liquid cell solution can be displaced from the bottom of the funnel by pumping. The cell suspension is seeded only at the bottom of the well, inside 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 is then transferred to an incubator to promote cell growth and proliferation. In some embodiments, the concentrator mask remains on the well plate during incubation. In other embodiments, the concentrator mask may be removed from the well plate prior to incubation. In additional embodiments, the concentrator mask remains in the well plate during culture, preventing the seeded cells from In certain embodiments of the present technology, cells are removed from the wells near the edges of the well walls. A region of concentrated cells is seeded in the center of the well while none are present at the periphery of the well.

[0049] In some embodiments, the distal elastomeric portion of the second open end is in the form of an O-ring. Compliant seal materials include resilient, essentially fluid-impermeable materials. The compliant seal material can be of any suitable shape to fit the edge of the second open end. For example, compliant seal materials are used in toroidal shaped O-rings, rectangular cross-sections, a gasket, metal gasket, or another type of compliant material having In one embodiment, the compliant seal material forms a fluid seal with the opposing well surfaces. In another embodiment, the material may be a fluoroelastomer or other material that forms a hole. The compliant seal material is silicone rubber. The client seal material creates a radial seal between the second open end and the well surface. It is contemplated that other seal orientations may also be used. Depending on the temperature and other cell culture medium components and conditions, various rubbers, e.g., fluoropolymers, , Buna, EPDM, or in some cases, compliant overplating The compliant seal material can be metallic with a gusset. If possible, coat with a chemically inert and biologically compatible coating. You can also use it.

[0050] In some embodiments, the concentrator mask of the present technology is adapted to mate with a flat-bottom cell culture well. In other embodiments, the concentrator mask of the present technology may be configured as follows: It is configured to interface with a dimple well such as an XF well.

[0051] In yet another embodiment, the concentrator mask of the present technology includes a continuous rib molded into the bottom of the well. The well is configured to interface with the well of the present technology, including the casing.

[0052] Therefore, the concentrator mask of this technology allows for the deposition of small particles in the central cell seeding area within the larger well. In some embodiments, the concentrator mask funnel can be used in a method for seeding cells. is used in a manner that concentrates cell seeding in the center of the bottom of the well. The concentrator mask seeds the majority of cells above the bottom area of ​​the central well at the desired cell concentration. In other embodiments, the concentrator mask of the present technology is a region at the bottom of the well where cells are not desired. can be used in a manner that eliminates cell seeding from

[0053] In some embodiments, the cell seeding concentration mask of the present technology allows cells in suspension to be concentrated on the well surface. with a cell suspension in combination with centrifugation and surface coating to allow adhesion to the surface. In certain embodiments, the concentrator mask may be removed prior to downstream analysis. It is possible.

[0054] The method may further include analysis of the cells, such as analysis by an Agilent XF assay. This can be done.

[0055] In some methods of the present technology, the method comprises removing the concentrator mass by a pipetting action or the like. The method further includes removing air trapped at the bottom of the well / well interface.

[0056] In some embodiments, about 5.0 μl to about 20 μl of medium is pipetted into the concentrator mask. In another embodiment, about 10 μl to about 15 μl of concentrated medium can be added. In some embodiments, about 12.5 μl can be pipetted into the instrument mask. The media can be pipetted into the concentrator mask. The method can be performed using volumes that do not introduce significant pipetting errors.

[0057] In some embodiments of the present technology, the seeded multi-well plates are In some of these embodiments, the concentrator mask may be It can remain in place during cell culture. Thus, certain embodiments of the present technology include: Compatible with poorly adherent cells. Another embodiment of the technology relates to the culture of highly adherent cells.

[0058] In other embodiments, the concentrator mask and method of using same are manufactured by Agilent XF In an additional embodiment, the plate is compatible with commercially available multi-well plates, such as well plates. , the concentrator mask and method of using same includes a continuous ring on the inner bottom well surface Compatible with the latest technology multi-well plates.

[0059] Figure 2A is a schematic diagram of a concentrator mask for seeding cells into multiwell plates. In this embodiment, the concentrator mask 201 has a frame surface 202 and a frame side 203. In this embodiment, the frame is also defined by the multi-well plate. The concentrator mask includes frame tabs 204 for alignment and stacking of the concentrator masks onto the frame. 205 extends from the frame. The funnel has a first open end 206 and a second open end 207. Included, 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 the funnel 205, a liquid-tight interface is created between the bottom of the well and the distal end 208 of the funnel. FIG. 2B is a cross-sectional schematic diagram of FIG. 2A, showing the micro-organisms inserted into the wells of a multi-well plate. 1 shows a cross section of a concentrator mask for seeding cells.

[0060] The concentrator mask of this technology is compatible with standardized multi-well plates and in any manner or orientation, including having the appropriate number and spacing of funnels to fit For example, in some embodiments, the concentrator mask of the present technology can be configured to In other embodiments, the concentrator mask of the present technology includes at least 1 funnel. In other embodiments, the concentrator mask of the present technology includes at least 24 funnels. In some embodiments, the concentrator mask of the present technology has at least 48 funnels. In other embodiments, the concentrator mask of the present technology includes at least 96 funnels. In other embodiments, the concentrator mask of the present technology includes at least 384 funnels. In an embodiment, the concentrator mask of the present technology includes at least 1536 funnels.

[0061] In some embodiments, the concentrator mask of the present technology has a thickness of about 0.5 mm to about 6.0 mm. In some other embodiments, the concentration of the present technology is controlled by a funnel having an inner diameter. The mask 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 has 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 a diameter of about 2 It is configured to contain a funnel having an inner diameter of 0.0 mm.

[0062] [Cell seeding system including multi-well plate and concentrator mask] This technology can also be combined with continuous ring multiwell plates as described herein. a cell seeding system including a concentrator mask as described herein for use in conjunction with In this embodiment, the multiple funnels of the concentrator mask correspond to the multiple funnels of the multi-well plate. The well is configured to fit into the well.

[0063] The cell seeding system of the present technology comprises a frame surface, a frame side surface extending from the frame surface, and and a frame having a well, each well having an open end, a closed end opposite the open end, and a well having a closed end opposite the open end. a plurality of wells having at least one wall between the ends, the open end of each well comprising: The closed end is surrounded by a frame surface and has a well surface that is between the walls and in contact with the walls. a plurality of wells, each of the wells having at least one continuous ring on the well surface at the closed end thereof; and a multi-well plate comprising:

[0064] The cell seeding system also has a frame surface and a frame side extending from the frame surface. a frame having a plurality of funnels extending from a surface of the frame, each funnel having a first open end; and a plurality of funnels having a first open end and a second open end. is connected to the frame surface and has a larger diameter than the second open end.

[0065] In the cell seeding system of the present technology, the multiple funnels of the concentrator mask are Multiwell plate so that the closed end of each well makes contact with the continuous ring on the well surface. In some embodiments, the bottom portion of the distal end is configured to fit into a plurality of wells. In some embodiments, the side portion of the distal end contacts the top of the continuous ring. Contact with the side of the ring.

[0066] FIG. 3 shows a multi-well plate having wells containing a continuous ring on the well surface at the closed end of the well. 1 is a cross-sectional schematic diagram of a cell seeding system including a concentrator mask inserted into a cell plate. In this embodiment, the cell seeding system 301 is configured such that the funnel of the concentrator mask 201 is a multi-well Concentrator mixers configured to fit together to be inserted into the wells of plate 101 The cell seeding system 3 is formed by the disk 201 and the multi-well plate 101. 01 includes a multi-well frame surface 102, a multi-well frame side surface 103, and a multi-well frame. The multiwell frame includes a multiwell frame base 104. In this embodiment, the multiwell frame , also includes multi-well frame tabs 105 for plate handling. The well walls 106 (not visible) define the well, and in this embodiment, the surrounding well pads. The multi-well plate wells also include a continuous ring 109 The continuous ring 109 includes an open end (not visible) and a closed end 108. A cell seeding area 110 is formed on the central portion of the edge 108 .

[0067] The concentrator mask 201 of the cell seeding system 301 comprises a frame surface 202 and a frame The concentrator mask also includes a plurality of funnels 205. The funnels have a first opening The concentrator mask 201 includes an open end 206 and a second open end 207. The end 207 is configured to be inserted into the well of the multi-well plate 101. In an embodiment, the second open end 207 of the funnel is a continuation of the closed end 108 of the well on the well surface. The second open end 20 is configured to contact the ring 109. In some embodiments, the second open end 20 7 includes a distal portion 208. In some embodiments, the second open end 207 and the continuous ring The interface between the second open end 2 and the lug 109 creates a fluid-tight seal. The interface between the 07 and the continuous ring 109 does not form a fluid-tight seal. It also includes multi-well plate frame tabs 105 for plate handling. The system also aligns and inserts the concentrator mask 201 into the multiwell plate 101. The concentrator includes a mask frame tab 204 for attaching the mask to the concentrator.

[0068] This technique also allows for seeding cells in the center of the wells of a multi-well plate. In some embodiments, the method includes: adding a liquid medium containing the cells to a first open funnel end 206 of the cell seeding system, As a result, the cells form at least one continuous ring 10 on the surface of the well at the closed end 108 of the well. 9. In some embodiments, the seeding The assembled cell seeding system 301 can be transferred to an incubator for cell growth. In this embodiment, the concentrator mask 201 can be removed from the multiwell plate 101. In another embodiment, the concentrator mask 201 can be a multi- It may remain inserted in the well plate 201. In some embodiments, the concentrator may The mask 201 is removed from the multi-well plate 101 prior to incubation. The concentrator mask 201 is placed in the multi-well plate while the cells are being cultured for growth. In some embodiments, the concentrator mask 201 may remain inserted in the port 101. , can be removed from the multiwell plate 101 prior to analysis of the cells. In this embodiment, the concentrator mask 201 remains on the multiwell plate 101 during cell analysis. In some embodiments, analyzing the cells comprises optical reading.

[0069] The technology is available in 6, 12, 24, 48, 96, 384, and 1536 well formats. Figure 4 shows a schematic diagram of the 96-well plate of this technology. In this embodiment, the multi-well has a well that includes a continuous ring on the inside bottom surface of the well. The plate 401 is connected to the frame surface 102, the frame side 103, and the frame base 104. The multiwell plate 401 is defined by a frame having well walls 104. 06, an open end 107, and a closed end 108 (not visible). The closed end 108 of the well The micro-organism further includes a continuous ring 109 that defines a cell seeding area 110. The multi-well plate 401 is a peripheral well plate moat or foil for plate processing. Although the multi-well plates of the present technology do not include frame tabs, in other embodiments, the multi-well plates of the present technology may include Includes peripheral well plate moats and frame tabs for

[0070] [Metabolic measurement] In some embodiments, the methods, devices, and systems are directed to the area of ​​microrespirometry. These include respiration measurements performed in whole animals and other methods for measuring cell biology. In contrast, quantitative measurements of the bioenergetic or metabolic state of a small number of cells are possible. In the past, microrespirometry was performed using milliliters of cell membranes to measure cellular metabolism. This was performed using a microscopic glass flow cell utilizing cell culture and a Clark electrode. The technique is not microscopic, easy, or high-throughput. Seahorse Bios Science flux analyzers and assays are available for 8, 24, and 96 plastic cells. Microrespirometry by introducing a comprehensive assay that can be easily performed in culture plates The resulting technology allows for a wide range of stimulants, inhibitors, and custom By introducing drugs and measuring the changes in oxygen consumption and proton production, the glycolytic pathway This allows for the complex characterization of both the oxidative phosphorylation and oxidative phosphorylation pathways. Additional details are provided in U.S. Patent Application Serial No. 15 / 896,255, which is incorporated by reference. and is hereby incorporated by reference in its entirety.

[0071] In another aspect of the invention, the methods, devices, and systems provide for the detection of individual cell types in culture. In some embodiments, the methods, devices, and systems are used for metabolic measurements. The system described herein can be used to analyze cells in contact co-culture. For example, the multi-well plates described herein may be used in The well plate contains a first cell type arranged inside the cell seeding area surrounded by a continuous ring. by placing a second cell type between the continuous ring and the well wall, outside the continuous ring. In some embodiments, the non-contact co-culture can be maintained by The different cell types in a non-contact co-culture are in fluid communication with each other. The different cell types in a contact co-culture are not in fluid communication with each other.

[0072] An additional aspect of the present invention includes a plurality of consecutive rings of any shape, the consecutive rings being of different shapes. Preparation and characterization of non-contact co-cultures by seeding cells into different cell seeding areas / segments Multiple cell-seeded areas or segments of any size, shape or configuration for cell growth and maintenance Furthermore, certain aspects of the present technology provide a grid of cell seeding regions rather than a ring. or alternative configurations, including shaped physical barriers on the well surface. The non-contact co-culture of this technology allows different cell types to be seeded in different cell seeding areas. If generated by the method, device, and system, one cell at a given time can be generated. Metabolic measurements can be taken independently from the seeded area / segment. Using the method's equipment and system, two or more or all cell seeding areas can be cultured at a given time. Metabolic measurements can be obtained from the region / segment. Details are provided in U.S. Patent Application Serial No. 15 / 896,255, which is incorporated herein by reference. is incorporated herein in its entirety.

[0073] The methods, devices, and systems use one or more sensors to measure the viability of a cell population. The sensors can measure the physical properties of the sample. sensors, surface plasmon resonance sensors, sensors based on the optical diffraction principle, sensors based on Wood's anomaly principle The sensor may be a cellular sensor, an acoustic sensor, or a microwave sensor. The method is not limited to measurements or sensors, but may instead be combined with any desired cell analysis approach. The present system, apparatus, and method may be used in combination by those skilled in the art. configured to measure one or more properties of the sample in the well as described herein. The sensor may include one or more of the aforementioned sensors.

[0074] The methods, devices and systems can be used in a variety of fields related to cell culture and analysis. Such fields include biological research, drug discovery, and clinical diagnostics, but For example, this device can be used as a drug discovery tool, for example, for co-culture, Effects on cellular metabolism, such as protein secretion or intracellular / extracellular ion exchange. A variety of molecules can be screened. The present methods, devices and systems also Determine the health of cells in culture, including co-cultures, both before and after the following assays are performed. These can be used to determine the activity of the target protein, thereby improving the performance of such assays.

[0075] [Cell population] The cell populations used in the present methods and devices can include any cell of interest. Cells include bacteria, fungi, yeast, prokaryotic cells, eukaryotic cells, animal cells, human cells, and / or At least some of the cells may be derived from the surface of blood vessels. At least a portion of the cells may be suspended in the medium. At least a portion of the tissue may comprise a living tissue, an organoid, a spheroid, or a cultured tissue. In some embodiments, at least a portion of the cells adhere to the closed end or wall of the well. is doing.

[0076] Known cell lines can be used as cell types in the present methods, devices, and systems. For example, known cell lines that can be used in combination with this technology include C8161, CC RF-CEM, MOLT, mIMCD-3, NHDF, HeLa, HeLa-S3, Hu hl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPa Cell, 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, W EHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bc l-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1 , COS-6, COS-M6A, BS-C-1 monkey kidney epithelium, BALB / 3T3 mouse embryo 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, B 16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR2 93. 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-L2 3 / R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EM T6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, H EK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, J urkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYOl, L NCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-1OA, MDA -MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MD CK II, MOR / 0.2R, MONO-MAC6, MTD-1A, MyEnd, NC I-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI- H69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT Cell line, Peer, PNT-1A / PNT2, RenCa, RIN-5F, RMA / RMA S, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cells 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 (e.g., See American Type Culture Collection (ATCC), Manassas, Virginia. These or other cell lines may be used in the present methods and devices as a first cell type or a second cell type. In some embodiments, the first cell type can be a cell of interest. the second cell type is a known cell line; .

[0077] Exemplary Embodiments 1. A multiwell plate for cell populations in liquid medium, comprising: a frame having a frame surface and a frame side extending from the frame surface; Each well has an open end, a closed end opposite the open end, and at least one space between the open end and the closed end. a plurality of wells each having at least one wall, the open end of each well being bounded by a frame surface; The closed end is between and in contact with at least one wall. a plurality of wells, each well surface including a well surface having a plurality of wells; at least one continuous ring on the well surface at the closed end of one or more wells; Included are multi-well plates. 2. At least one continuous ring defines at least one cell seeding area on the well surface. The assay chamber is configured to define a cavity that cooperates with a lid, plunger, or another element to define an assay chamber. 2. The multi-well plate of embodiment 1, in which microchambers can be formed. 3. The multiwell plate includes multiple wells configured to define multiple cell seeding areas. 2. The multi-well plate of embodiment 1, comprising a continuous ring. 4. At least one continuous ring has a height of about 0.01 mm to about 2 mm. The multi-well plate of any of the preceding embodiments. 5. At least one continuous ring has an inner diameter of about 0.5 mm to about 6.0 mm, e.g., 10. The multi-well plate of any of the preceding embodiments, having an inner diameter of about 2.0 mm. 6. A concentrator mask for seeding cells in liquid medium into multi-well plates. , a frame having a frame surface and a frame side 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; a funnel having a distal end; The first open end is connected to the frame surface and has a larger diameter than the second open end. A concentrator mask. 7. The concentrator mask of embodiment 6, wherein the second open end comprises a distal elastomeric portion. 8. The concentrator mask of embodiment 6 or 7, wherein the concentrator mask includes at least 8 funnels. nine. 9. The funnel has an inner diameter of about 0.5 mm to about 6.0 mm, for example, an inner diameter of about 2.0 mm. 9. The concentrator mask of embodiments 6 to 8. 10. A method for seeding cells in the central portion of a culture well, comprising: 6. The method of claim 1, wherein the cells are placed in at least one cell-seeding region on the well surface. adding a liquid medium comprising: 11. The culture well is transferred to an incubator and cultured after seeding the cells, embodiment 1 0 ways. 12. The cultured cells are substantially free of incubator-induced edge effects. 12. The method of embodiment 11, wherein the method does not include or does not include any of the above steps. 13. A cell seeding system comprising a multiwell plate and a concentrator mask, The multi-well plate has a frame surface and a frame side extending from the frame surface. Each well has an open end, a closed end opposite the open end, and a pair of open and closed ends. a plurality of wells having at least one wall therebetween, the open end of each well being the closed end is surrounded by a well surface between the walls and in contact with the walls. a plurality of wells, and at least one continuous ring on the well surface at the closed end of each well; Including, The concentrator mask has a frame surface and a frame side extending from the frame surface. a frame; 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 is connected to the frame surface and the second open end is a funnel having a large diameter; The plurality of funnels are configured to fit into the plurality of wells, such that the second opening of the funnels The edge forms an interface with a continuous ring on the well surface at the closed end of one or more wells. , cell seeding system. 14. The interface between the second open end and the continuous ring is small enough to reduce the spreading of the liquid. A system according to embodiment 13, wherein the gap is small. 15. The system of embodiment 13, wherein the second open end comprises a distal elastomeric portion. 16. The interface is physical contact between the distal elastomeric portion of the second open end and the continuous ring. 16. The system of embodiment 15, wherein the fluid-tight seal is formed. 17. A method for seeding cells in a multiwell plate, comprising: Adding a liquid medium containing cells to a first open end of the cell seeding system of embodiment 13. and wherein the cells are arranged in an area surrounded by at least one continuous ring on the surface of the well. To settle, a way. 18. After seeding the cells, the multi-well plate is transferred to an incubator, embodiment 1 7. The cultured cells showed no incubator-induced edge effect. It is considered to be free or substantially free. 19. Before placing the multiwell plate in the incubator, remove the concentrator mask from the multiwell plate. 20. The method of embodiment 18, wherein the well plate is removed. 20. Further analysis of cells in multiwell plates to obtain optical measurements. 18. The method of embodiment 17, comprising:

[0078] [Example 1] Cells were seeded, grown, and analyzed in multiwell plates containing continuous rings of this technology. These cells were seeded and expanded in multiwell plates containing standard wells without continuous rings. The continuous rings tested had a wall height of 0.2 mm and an inner diameter of 1.5 mm. The same number of cells (4,500) was analyzed in both well types.

[0079] After seeding and growing the cells, the oxygen consumption rate ( The speed was measured by taking optical measurements. The number of cells (4,500) was significantly higher than that of cells cultured in standard wells without a continuous ring. and found that they produced a three-fold higher signal intensity when cultured in wells containing continuous rings. showed.

[0080] It should be understood that in light of the present disclosure, methods and apparatuses can be implemented in accordance with the present teachings. Furthermore, various components, materials, structures, and parameters are provided for illustrative purposes only. It is included as a guide only and not as a limitation. The present invention may be implemented in other applications and may involve the use of components, materials, structures, etc. to implement these applications. The structure and material may be determined while remaining within the scope of the appended claims.

Claims

1. A multiwell plate to be used in combination with a concentrator mask, The concentrator mask comprises 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, The aforementioned multiwell plate 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, At least one continuous ring is provided on the well surface of the closed end of each of the wells, Includes, A multiwell plate in which 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.

2. The multiwell plate according to claim 1, wherein the at least one continuous ring is configured to define at least one cell seeding region on the well surface.

3. The multiwell plate according to claim 1, wherein the multiwell plate includes a plurality of continuous rings configured to define a plurality of cell seeding regions.

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

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

6. A method for seeding cells in the central portion of a culture well, A method comprising adding a liquid culture medium containing cells to the at least one cell seeding region on the well surface of claim 2.

7. The method according to claim 6, further comprising culturing the culture wells after seeding the cells.

8. The method according to claim 7, wherein the cultured cells are substantially free from the edge effect induced by the incubator.

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