EP4623064A1

EP4623064A1 - Microplates with retention structure and method of using the same

Info

Publication number: EP4623064A1
Application number: EP23821759.0A
Authority: EP
Inventors: Feng Li; Gregory Roger Martin; Sweta Nimesh PARIKH; Hilary Anne SHERMAN
Original assignee: Corning Inc
Current assignee: Corning Inc
Priority date: 2022-11-21
Filing date: 2023-11-03
Publication date: 2025-10-01
Also published as: JP2026504643A; CN120225651A; WO2024112417A1

Abstract

A microplate including a plurality of wells for receiving a dome of a supporting matrix, each well of the plurality of wells comprising a bottom surface having at least one retention structure to provide mechanical retention of one of the assay samples, the at least one retention structure comprising a diameter ranging between about 0.5 millimeters and about 5 millimeters and a height, and wherein a ratio of the height to the diameter ranges from about 10% to about 30% and a method of using the same.

Description

MICROPLATES WITH RETENTION STRUCTURE AND METHOD OF USING THE SAME

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application Serial No. 63/426845 filed on November 21, 2022, the content of which is relied upon and incorporated herein by reference in its entirety.

FIELD

[0002] The present disclosure relates to microplates. More specifically, the present disclosure relates to microplates having at least one retention structure within a well for providing mechanical retention of a dome of a supporting matrix having suspended cells.

BACKGROUND

[0003] Although two-dimensional cell cultures, which provide a flat monolayer of cells, have helped researchers gain substantial knowledge and understanding of cell behavior, cell function and cell differentiation, researchers have observed that two-dimensional cell cultures provide markedly different behavior in cell polarity, stem cell differentiation, migration, gene expression, and tissue organization. To create a cell environment that more closely mimics the in vivo cell environment, researchers use three-dimensional scaffolds to develop organoids. A supporting matrix, for example, a hydrogel, is used as the three-dimensional scaffold in the organoid culture dome method.

[0004] The present disclosure relates to technology for three-dimensional organoid culture dome method. The organoid culture dome method is an established procedure for forming dome structures in wells of microplates using a supporting matrix. While three-dimensional dome structures are commonly used in organoid models, there are several drawbacks to the organoid culture dome method. First, forming three-dimensional dome structures within a well of a microplate requires extensive training and practice by an individual, and even still, results can be variable depending on the skill level of the individual. For example, the sizes of the dome structures can vary significantly since the individual is required to quickly disperse droplets into the wells to avoid gelation of the supporting matrix during the process. Having size variations of domes is not optimal for organoid culture consistency. Second, the domes are typically randomly placed when positioning multiple domes within a well. If the domes are placed too closely to each other, then dome fusion can occur due to the proximity of the neighboring domes. Moreover, a dome can collapse if an individual places the dome too close to the sidewall of a microplate well. Yet another common problem with the formation of dome structures is the loss of domes during the medium exchange process as a result of medium seeping between the domes and the surface of the microplate well.

SUMMARY

[0005] In view of the foregoing, the aforementioned challenges may be at least partially addressed by providing at least one retention structure within a well of a microplate for providing mechanical retention of a dome of a supporting matrix.

[0006] An exemplary embodiment of the present disclosure provides a microplate comprising a a plurality of wells for receiving a dome of a supporting matrix, each well of the plurality of wells comprising a bottom surface comprising at least one retention structure to provide mechanical retention of one of the assay samples, wherein the at least one retention structure may comprise a diameter ranging between about 0.5 millimeters and about 5 millimeters and a height, and wherein a ratio of the height to the diameter may range from about 10% to about 30%.

[0007] In some embodiments each well of the plurality of wells may comprise a plurality of retention structures configured to receive one of the assay samples.

[0008] In some embodiments, the at least one retention structure may comprise a continuous wall, and the height of the at least one retention structure may range between about 1 millimeter and about 3 millimeters. Further, in some embodiments, the continuous wall may be configured to retain a volume of one of the assay samples deposited within the continuous wall ranging between about 5 microliters and about 50 microliters.

[0009] In other embodiments, the at least one retention structure may comprise a plurality of discontinuous walls, and the height of the at least one retention structure may range between about 1 millimeter and about 3 millimeters. Further, in some embodiments, the discontinuous walls may be configured to retain a volume of one of the assay samples deposited within the discontinuous walls ranging between about 5 microliters and about 50 microliters.

[0010] In other embodiments, the at least one retention structure may comprise a plurality of radially spaced apart posts. Each post may comprise a height ranging between about 1 millimeter and about 3 millimeters. The plurality of radially spaced apart posts, in some embodiments, may be configured to retain a volume of one of the assay samples deposited within the radially spaced apart posts ranging between about 5 microliters and about 50 microliters.

[0011] In some embodiments, each well of the plurality of wells may include a first retention structure centrally positioned on the bottom surface and in fluid communication with a set of second retention structures positioned on the bottom surface and spaced from the first retention structure. Each well may include a plurality of fluidic channels extending between the first retention structure and the set of second retention structures to provide fluid communication therebetween, each of the fluidic channels comprising an inlet for receiving cells suspended in the supporting matrix from the first retention structure and an outlet for delivering cells suspended in the supporting matrix to one of the second retention structures in the set of second retention structures.

[0012] In accordance with some embodiments, the plurality of wells may be arranged in rectilinear arrays of rows and columns to form a matrix, and each well of the plurality of wells may be a flat bottom well. The matrix may comprise, for example, a total of 6 wells, 12 wells, 24 wells, or 48 wells.

[0013] In some embodiments, the thickness of the bottom wall of the microplate may range between about 0.05 millimeters and about 1 millimeters, and the at least one retention structure may be formed by injection molding, hot pin embossing, or casting. The at least one retention structure, in some embodiments, may include a top edge formed with one of a beveled edge, a rounded edge, or a chamfered edge. The at least one retention structure in some embodiments may be optically transmissive. [0014] Another exemplary embodiment of the present disclosure provides a method of using a microplate comprising providing a microplate comprising a plurality of wells arranged in rectilinear arrays of rows and columns for receiving assay samples of suspended cells in a supporting matrix, each well of the plurality of wells comprising a bottom surface comprising a plurality of retention structures to provide mechanical retention of one of the assay samples within each retention structure, wherein each retention structure may comprise a diameter ranging between about 0.5 millimeters and about 5 millimeters and a height, and wherein a ratio of the height to the diameter may range from about 10% to about 30%; and depositing an assay sample within each of the retention structures.

[0015] The method, in some embodiments, may comprise incubating the supporting matrix within each of the retention structures to polymerize the supporting matrix, and overlaying the supporting matrix with media containing niche factors to form organoids in the supporting matrix retained within each of the retention structures.

[0016] In some embodiments, the method may further comprise imaging the organoids. In some embodiments, the method may include performing an experiment using the organoids in the supporting matrix retained within each of the retention structures. Further, in some embodiments, the method may comprise a step of warming the microplate before the assay sample is deposited within each of the retention structures.

[0017] Another exemplary embodiment of the present disclosure comprises a unitary molded microplate comprising (i) a rectangular frame including a planar surface and sidewalls; (ii) a plurality of wells within the planar surface, each well of the plurality of wells comprising a bottom; and (iii) a plurality of optically transmissive retention structures protruding from the bottom of each well of the plurality of wells, each optically-transmissive retention structure may be configured to mechanically retain an assay sample of suspended cells in a supporting matrix, wherein each optically transmissive retention structure comprises a diameter that may range between about 0.5 millimeters and about 5 millimeters and a height, and wherein a ratio of the height to the diameter may range from about 10% to about 30%. In some embodiments, the plurality of optically transmissive retention structures was formed by molding recesses in the bottom of each of the plurality of wells. In another embodiment, the plurality of optically transmissive retention structures was formed by hot pin embossing recesses into the bottom of each well of the plurality of wells.

[0018] The following will describe embodiments of the present disclosure. However, the present disclosure is not limited to the described embodiments and various modifications of the disclosure are possible without departing from the basic principles described herein.

BRIEF DESCRIPTION OF THE DRAWING(S)

[0019] Various embodiments are disclosed, by way of example only, with reference to the accompanying schematic drawings below in which corresponding reference symbols indicate corresponding parts. Like drawing numbers on different drawing views identify identical, or functionally similar, structural elements.

[0020] FIG. 1 is a top plan view of an exemplary microplate having retention structures within a plurality of wells;

[0021] FIG. 2 is a partial top plan view of the exemplary microplate shown in FIG. 1;

[0022] FIG. 3 is a cross-sectional view of a plurality of wells of the exemplary microplate taken from lines 3-3 of FIG. 3;

[0023] FIG. 4 is a cross-sectional view of a plurality of wells of the exemplary microplate taken from lines 4-4 of FIG. 3 ;

[0024] FIG. 5 is a cross-sectional view of one of the retention structures shown in FIGS. 3 and 4 including a partial view of a continuous wall and having a dome of supporting matrix;

[0025] FIG. 6 is a cross-sectional view of one of the retention structures shown in FIGS. 3 and 4, except showing discontinuous walls;

[0026] FIG. 7 is a cross-sectional view of one of the retention structures shown in FIGS. 3 and 4, except showing a cavity without a wall protruding from the bottom surface of the well and having a dome of supporting matrix; [0027] FIG. 8 is a perspective view of a well of an exemplary microplate having sets of posts forming each of the retention structures;

[0028] FIG. 9 is a cross-sectional view of the retention structure shown in FIGS. 3 and 4, except without a cavity and with the retention structure having a beveled edge;

[0029] FIG. 10 is a top plan view of an exemplary microplate having retention structures and showing channels fluidly connecting the retention structures;

[0030] FIG. 11 A is a cross-sectional view of one of the retention structures within one of the wells of the exemplary microplate taken from lines 11-11 of FIG. 10, showing hot pin embossing forming a cavity;

[0031] FIG. 1 IB is a cross-sectional view of the retention structure shown in FIG. 11A after the hot pin embossing; and

[0032] FIG. 12 is a flow chart of a method of using the microplate in accordance with an exemplary embodiment of the present disclosure.

DETAILED DESCRIPTION

[0033] The present disclosure is not limited to the particular methodology, materials and modifications described and as such may, of course, vary. Moreover, the terminology used herein is for the purpose of describing particular aspects only and is not intended to limit the scope of the claims.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure pertains. Methods, devices, or materials similar or equivalent to those described herein can be used in the practice or testing of the example embodiments.

[0035] As used herein, the term “substantially” is synonymous with terms such as “nearly,” “very nearly,” “about,” “approximately,” “around,” “bordering on,” “close to,” “essentially,” “in the neighborhood of,” “in the vicinity of,” etc., and such terms may be used interchangeably as appearing in the specification and claims. It should be appreciated that the term “proximate” is synonymous with terms such as “nearby,” “close,” “adjacent,” “neighboring,” “immediate,” “adjoining,” etc., and such terms may be used interchangeably as appearing in the specification and claims. The term “approximately” is intended to mean values within ten percent of the specified value. The term “about” and its synonymous terms mean that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. When the term “about” is used in describing a value or an end point of a range, the disclosure should be understood to include the specific value or end point referred to. Whether or not a numerical value or endpoint of a range in the specification recites “about,” the numerical value or end point of a range is intended to include two embodiments: one modified by “about,” and one not modified by “about.” It will be further understood that the end points of each of the ranges are significant both in relation to the other end point, and independently of the other endpoint.

[0036] The use of “or” in the present application is with respect to a “non-exclusive” arrangement, unless stated otherwise. For example, when stating that “item x is A or B,” it is understood that this can mean one of the following: (1) item x is only one or the other of A and B; (2) item x is both A and B. Alternately stated, the word “or” is not used to define an “exclusive or” arrangement. For example, an “exclusive or” arrangement for the statement “item x is A or B” would require that x can be only one of A and B. Furthermore, as used herein, “and/or” is intended to mean a grammatical conjunction used to indicate that one or more of the elements or conditions recited may be included or occur. For example, a device comprising a first element, a second element and/or a third element, is intended to be construed as any one of the following structural arrangements: a device comprising a first element; a device comprising a second element; a device comprising a third element; a device comprising a first element and a second element; a device comprising a first element and a third element; a device comprising a first element, a second element and a third element; or, a device comprising a second element and a third element.

[0037] Moreover, as used herein, the phrases “comprises at least one of’ and “comprising at least one of’ in combination with a system or element is intended to mean that the system or element includes one or more of the elements listed after the phrase. For example, a device comprising at least one of: a first element; a second element; and, a third element, is intended to be construed as any one of the following structural arrangements: a device comprising a first element; a device comprising a second element; a device comprising a third element; a device comprising a first element and a second element; a device comprising a first element and a third element; a device comprising a first element, a second element and a third element; or, a device comprising a second element and a third element. A similar interpretation is intended when the phrase “used in at least one of” is used herein.

[0038] References herein to the positions of elements (e.g., “top,” “bottom,” “above,” “below,” etc.) are merely used to describe the orientation of various elements in the figures. The orientation of various elements may differ according to other exemplary embodiments, and such variations are intended to be encompassed by the present disclosure.

[0039] Specific and preferred values disclosed for components, ingredients, additives, dimensions, conditions, times, and like aspects, and ranges thereof, are for illustration only; they do not exclude other defined values or other values within defined ranges. The compositions, articles, and methods of the disclosure can include any value, or any combination of the values, specific values, more specific values, and preferred values described herein, including explicit or implicit intermediate values and ranges.

[0040] In the present disclosure, a microplate having retention structures and methods of using the same are described. The retention structures can provide control for positioning organoid domes within wells of a microplate. Having at least one retention structure within a well of a microplate may allow more even distribution of organoid domes within a well and, thus, may allow nutrients from the media to be used more evenly among the domes during the culture. It can also help standardize and improve the quality of the organoid culture. Moreover, the microplate having retention structures can provide better control for the organoid dome sizes, especially with manual operation by an individual. Further, the retention structures can provide physical support and protection to the organoid domes and reduce the loss of organoid domes during operational procedures such as medium exchange or drug treatment.

[0041] Turning now to the figures, FIGS. 1-4 show various views of a microplate 100 having a plurality of wells 102. The microplate 100 in various embodiments may be a unitary molded microplate having a deck 104 formed of a rectangular frame 106 comprising a planar surface 108, sidewalls 110, and a bottom wall 112. Each of the plurality of wells 102 of the microplate 100 further includes well walls 114 and a bottom 116 having a bottom surface 118. In the illustrated examples of FIGS. 3 and 4, the plurality of wells 102 may be cylindrical shaped, with a flat bottom 116. The deck 104 may support rectilinear arrays of rows and columns to form a matrix, although in further embodiments, other array configurations may be adopted. For example, as shown in FIG. 1, the deck 104 may support 24 wells. However, the deck 104 may support fewer or additional wells. For example, the microplate 100 may include a matrix comprising a total of 6 wells, 12 wells, 48 wells, or more. The deck 104 can be formed in other geometrical shapes depending on the desired number of wells and/or the desired use of the microplate 100. The microplate 100 may optionally include a microplate cover (not shown) and a microplate base (not shown).

[0042] The microplate 100 may be made of polymeric material. In some embodiments, the microplate 100 may comprise polystyrene. For example, the microplate 100 can be made of clear polystyrene, solid black polystyrene, or white polystyrene. In another embodiment, the microplate 100 may comprise an optically clear bottom with either a black opaque polystyrene microplate body or a white opaque polystyrene microplate body. The microplate 100 may alternatively be made of polycarbonate, polypropylene, poly vinyl chloride (PVC), polyethylene terephthalate, UV transparent material, glass material, cyclic olefin copolymer (COC), or a combination thereof. However, the microplate 100 may be made of other materials. The bottom 116 of each well 102 comprises a bottom surface 118 that can be treated or not treated. In some embodiments, the bottom surface 118 may be tissue culture treated or coated with poly-D-lysine (PDL), collagen, fibronectin, Corning® BioCoat™ surfaces, or Coming® PureCoat™ surfaces. Alternatively, in some embodiments, the bottom surface 118 is a Corning® CellBIND® Surface.

[0043] Each well 102 may comprise at least one retention structure 130 for mechanically retaining a supporting matrix as described below. In one embodiment, organoid cells may be mixed with a supporting matrix that provides a three-dimensional scaffold. In some embodiments, the organoid cells are mixed with a supporting matrix using the organoid culture dome method. Typically, the supporting matrix resembles an extracellular matrix, for example, a hydrogel that supports organoid growth and differentiation. An example supporting matrix that can be used is the Corning® Matrigel® matrix. In one example method, the supporting matrix may be placed on ice to maintain the supporting matrix at a low temperature of approximately 0°C and then a droplet having suspended cells ranging from about 5 microliters to about 50 microliters is dispersed within the boundary of a retention structure 130. The size of the retention structure 130 can be selected based on the desired size of the organoid domes as discussed in more detail below. Further, other supporting matrix materials can be used. Similarly sized droplets are disposed in neighboring retention structures 130 within a well 102 and within other wells 102. The microplate 100 may be pre- warmed in an incubator at about 36°C to about 40°C. In one embodiment, the incubator may be warmed to approximately 37°C before the droplets are dispersed on the microplate 100. In embodiments, the microplate 100 may be pre- warmed overnight. The size of the droplets may be determined at least in part by the number of wells as well as the height and diameter of the retention structure 130 within each well as discussed in more detail farther below. Once the cell and supporting matrix mixture are dispersed on the bottom surface 118 of each well 102, the supporting matrix may be incubated at 37° C to polymerize the gel and form a dome structure. In some embodiments, the supporting matrix may be gellified in about 5 to about 10 minutes, whereupon gellified material becomes a semisolid dome structure with organoid cells embedded in the structure. Media containing niche factors can be applied to the polymerized dome such that organoids form and enlarge. When used herein, the term “assay sample” refers to a droplet of cells in a supporting matrix that forms an organoid dome structure.

[0044] In embodiments, each well 102 may include a single retention structure 130. In some embodiments, each well may contain a plurality of retention structures 130. In certain embodiments, each well 102 may include between approximately 1 to 50 retention structures. For example, a well of a 24- well plate may comprise 5 to 7 retention structures 130 at the bottom 116 of each well, while a 6- well plate may comprise 20 to 30 retention structures 130 at the bottom 116 of each well. The retention structure 130 mechanically retains a droplet of cells suspended in a scaffolding material that resembles an extracellular matrix, also referred to as a supporting matrix, placed within a boundary defined by the retention structure 130, as described in more detail below.

[0045] As shown in FIGS. 3-7, each retention structure 130 can include a cavity 132 defined by walls 134 and a cavity bottom surface 128 and provides a three-dimensional region to retain a droplet of supporting matrix having suspended cells 180 that forms a dome structure. The size of the retention structure 130 and number of wells 102 of a microplate 100 can be selected based on the desired size of the organoid domes. As shown in FIGS. 3-6, in some embodiments, the retention structure 130 may comprise a raised wall 136 protruding from the bottom surface 118 of the well 102 and a cavity 132. The raised wall 136 and the bottom surface 118 of the well 102 may be integrally formed. In embodiments, the raised wall 136 may be a continuous wall 138 having a closed perimeter or circumference. For example, the retention structure 130 may be a closed circle, ellipse, polygon, or other shape. Each retention structure 130 includes a diameter (Di), and a height (Hi). The diameter (Di) is an inner diameter measured from the inner walls of the retention structures 130. The height (Hi) is measured from the cavity bottom surface 128 to a top surface 140 of the continuous wall 138. In embodiments, the diameter (Di) may range between about 0.5 millimeters and about 5 millimeters. For example, in some embodiments, the diameter (Di) may range between about 0.5 millimeters and 2.5 millimeters, between about 2.5 millimeters and 5.0 millimeters, between about 0.5 millimeters and 1.0 millimeters, between about 1.0 millimeter and about 1.5 millimeter, between about 1.5 millimeters and about 2 millimeters, between about 2 millimeters and about 2.5 millimeters, between about 2.5 millimeters and about 3.0 millimeters, between about 3.0 millimeters and about 3.5 millimeters, between about 3.5 millimeters and about 4.0 millimeters, between about 4.0 millimeters and about 4.5 millimeters, or between about 4.5 millimeters and about 5.0 millimeters, including all ranges and subranges therebetween.

[0046] In embodiments, the height (Hi) of the continuous wall 138 of the retention structures 130 may range between about 1 millimeter and about 3 millimeters. For example, in some embodiments, the height (Hi) may range between about 1 millimeter and about 1.5 millimeters, between about 1.5 millimeter and about 2 millimeters, between about 2 millimeters and about 2.5 millimeters, or between about 2.5 millimeters and about 3 millimeters, including all ranges and subranges therebetween. The height (Hi) and diameter (Di) can be selected based on the desired dome size to be formed from the dispensed droplet of cells suspended in the supporting matrix 180. That is, the retention structure 130 can be configured to hold a particular droplet size in a range of about 5 microliters to about 50 microliters.

[0047] For example, in some embodiments, each retention structure 130 can hold a droplet size of about 5 microliters. In other embodiments, each retention structure 130 can hold a droplet size of about 10 microliters. In yet other embodiments, each retention structure can hold a droplet size of about 50 microliters. In certain embodiments, the droplet size may range between about 5 microliters and about 10 microliters, between about 10 microliters and about 15 microliters, between about 15 microliters and about 20 microliters, between about 5 microliters and about 25 microliters, between about 20 microliters and about 25 microliters, between about 25 microliters and about 30 microliters, between about 30 microliters and about 35 microliters, between about 25 microliters and about 50 microliters, between about 35 microliters and about 40 microliters, between about 40 microliters and about 45 microliters, or between about 45 microliters and about 50 microliters, including all ranges and subranges therebetween.

[0048] Retention structure size can correspond to the number of wells 102 in each microplate 100 as described above. For example, 5 to 7 domes are usually generated with an average volume of about 5 to about 10 microliters and deposited on a 24-well plate. In some embodiments, a ratio of the height (H) to the diameter (D) may range from about 10% to about 30%. In some embodiments, the height (Hi) to diameter (Di) ratio may be about 10% to 12%, 10% to 14%, 10% to 16%, or 10% to 18%, from about 10% to about 20%, from about 12% to about 20%, from about 14% to about 20%, from about 16% to about 20%, from about 18% to about 20%, from about 20% to about 30%, from about 22% to about 30%, from about 24% to about 30%, from about 26% to about 30%, from about 28% to about 30%, including all ranges and subranges therebetween. In yet other embodiments, the height (Hi) to diameter (Di) ratio may be about 30%. As stated above, the continuous wall 138 of a retention structure 130 may be configured to retain a volume of one of the assay samples deposited within the continuous wall 138 ranging between about 5 microliters and about 50 microliters. However, the supporting matrix may expand in volume due to the absorption of culture media when the culture media is added to the dome culture. Thus, the volume of the organoid dome may increase to an expanded volume of 100 microliters. In some embodiments, the diameter of the organoid dome may be about 2 millimeters to about 4 millimeters. For example, the diameter of the organoid dome may be between about 2 millimeters and about 2.5 millimeters, between about 2.5 millimeters and about 3.0 millimeters, between about 3.0 millimeters and about 3.5 millimeters, or between about 3.5 millimeters and about 4.0 millimeters, including all ranges and subranges therebetween.

[0049] As shown in FIG. 6, in embodiments, the retention structure 130 may include a raised wall 136, wherein the raised wall 136 is a discontinuous wall or set of discontinuous walls 142. FIG. 6 illustrates a three-dimensional region to retain the droplet of supporting matrix having suspended cells 180 that forms a dome structure, but with the droplet 180 removed to show the discontinuous walls 142. The discontinuous walls 142 comprise an opening within the perimeter or circumference of the discontinuous walls. For example, the retention structure 130 can include a set of partial walls in the shape of a circle. Alternatively, the retention structure 130 can include a set of partial walls in the shape of an oval, polygon, or other shape. Each retention structure 130 includes a diameter (D2), and a height (H2). In embodiments, the diameter (D2) is an inner diameter measured from the inner walls of the retention structures 130. The height (H2) is measured from the cavity bottom surface 128 to a top surface 140 of the discontinuous walls 142. In embodiments, the diameter (D2) may range between about 0.5 millimeters and about 5 millimeters. For example, in embodiments, the diameter (D2) may range between about 0.5 millimeters and about 2.5 millimeters, between about 2.5 millimeters and about 5.0 millimeters, between about 0.5 millimeters and about 1.0 millimeters, between about 1.0 millimeter and about 1.5 millimeter, between about 1.5 millimeters and about 2 millimeters, between about 2 millimeters and about 2.5 millimeters, between about 2.5 millimeters and about 3.0 millimeters, between about 3.0 millimeters and about 3.5 millimeters, between about 3.5 millimeters and about 4.0 millimeters, between about 4.0 millimeters and about 4.5 millimeters, or between about 4.5 millimeters and about 5.0 millimeters, including all ranges and subranges therebetween.

[0050] In embodiments, the height of the discontinuous walls 142 of the retention structures 130 ranges may be between about 1 millimeter and about 3 millimeters. For example, in some embodiments, the height (H2) may range between about 1 millimeter and about 1.5 millimeters, between about 1.5 millimeter and about 2 millimeters, between about 2 millimeters and about 2.5 millimeters, or between about 2.5 millimeters and about 3 millimeters, including all ranges and subranges therebetween. The height (H2) and diameter (D2) can be selected based on the desired dome size formed from the dispensed droplet of cells suspended in the supporting matrix. A ratio of the height (H2) to the diameter (D2) may range from about 10% to about 30%, for example in a range from about 10% to about 12%, from about 10% to about 14%, from about 10% to about

16%, from about 10% to about 18%, from about 10% to about 20%, from about 12% to about

20%, from about 14% to about 20%, from about 16% to about 20%, from about 18% to about

20%, from about 20% to about 30%, from about 22% to about 30%, from about 24% to about

30%, from about 26% to about 30%, or from about 28% to about 30%, including all ranges and subranges therebetween. The discontinuous walls 142 of a retention structure 130 may be configured to retain a volume of one of the assay samples deposited within the discontinuous walls 142 ranging between about 5 microliters and about 50 microliters. However, the supporting matrix may expand in volume due to the absorption of culture media when the culture media is added to the dome culture. Thus, the volume of the organoid dome may increase to an expanded volume of 100 microliters or more.

[0051] In alternative embodiments, as illustrated in FIG. 7, the retention structure 130 may include the cavity 132 without a raised wall. In such embodiments, the cavity 132, defined by cavity walls 134 and a cavity bottom surface 128, provides a three-dimensional region to retain the droplet of supporting matrix having suspended cells 180 that forms a dome structure. In such embodiments, the retention structure 130 includes a diameter (D3), and a height (H3). In embodiments, the diameter (D3) is an inner diameter measured from the inner walls of the cavity 132. The height (H3) is measured from the cavity bottom surface 128 to the top of the cavity 132, which is the bottom surface 118 of the well 102. In embodiments, the diameter (D3) may range between about 0.5 millimeters and about 5 millimeters.

[0052] For example, in some embodiments, the diameter (D3) may range between about 0.5 millimeters and about 2.5 millimeters, between about 2.5 millimeters and about 5.0 millimeters, between about 0.5 millimeters and about 1.0 millimeters, between about 1.0 millimeter and about 1.5 millimeter, between about 1.5 millimeters and about 2 millimeters, between about 2 millimeters and about 2.5 millimeters, between about 2.5 millimeters and about 3.0 millimeters, between about 3.0 millimeters and about 3.5 millimeters, between about 3.5 millimeters and about 4.0 millimeters, between about 4.0 millimeters and about 4.5 millimeters, or between about 4.5 millimeters and about 5.0 millimeters, including all ranges and subranges therebetween.

[0053] In embodiments, the height of the cavity walls 134 of the retention structures 130 may range between about 0.5 millimeter and about 3 millimeters. For example, in embodiments, the height (H3) may range between about 1 millimeter and about 1.5 millimeters, between about 1.5 millimeter and about 2 millimeters, between about 2 millimeters and about 2.5 millimeters, between about 2.5 millimeters and about 3 millimeters, including all ranges and subranges therebetween. The height (H3) and diameter (D3) can be selected based on the desired dome size formed from the dispensed droplet of cells suspended in the supporting matrix. A ratio of the height (H3) to the diameter (D3) may range from about 5% to about 30%. The cavity 132 of a retention structure 130 may be configured to retain a volume of one of the droplets of supporting matrix having suspended cells 180 which forms a dome structure deposited within the cavity 132, wherein the volume may range between about 5 microliters and about 50 microliters. In certain embodiments, the volume range may be between about 5 microliters and about 10 microliters, between about 10 microliters and about 15 microliters, between about 15 microliters and about 20 microliters, between about 5 microliters and about 25 microliters, between about 20 microliters and about 25 microliters, between about 25 microliters and about 30 microliters, between about 30 microliters and about 35 microliters, between about 25 microliters and about 50 microliters, between about 35 microliters and about 40 microliters, between about 40 microliters and about 45 microliters, or between about 45 microliters and about 50 microliters, including all ranges and subranges therebetween. It should be appreciated, however, that the supporting matrix may expand in volume due to the absorption of culture media. Thus, the volume of the organoid dome may increase to an expanded volume of 100 microliters or more. For example, the organoid dome may increase to an expanded volume of between about 10 microliters to about 200 microliters, between about 10 microliters and about 50 microliters, between about 50 microliters and about 100 microliters, between about 100 microliters and about 150 microliters, or between about 150 microliters and about 200 microliters, including all ranges and subranges therebetween.

[0054] In other embodiments, as shown in FIG. 8, the retention structure 130 may comprise a plurality of radially spaced apart posts 150, each plurality of radially spaced apart posts 150 forming a set. It should be appreciated that each well 102 may comprise a plurality of retention structures 130. For example, in FIG. 8, seven (7) retention structures 130, each retention structure comprising a plurality of radially spaced apart posts 150, are shown. Each set of posts 150 forming a retention structure 130 may alternatively form an ellipse, polygon, or other shape. In embodiments, the posts 150 may each be cylindrical. However, other shapes are possible, including elliptical or polygonal, or including, but not limited to, triangular, square, or rectangular. In embodiments, the posts 150 of each retention structure 130 may surround a cavity, for example cavity 132, as shown in FIG. 7, within the bottom 116 of the well 102. In other embodiments, the posts 150 may be arranged on the bottom 116 of the well 102 without a cavity. The retention structure 130 of posts 150 may include 3 to 4 posts. However, additional posts 150 may be used to form a retention structure 130. Each retention structure 130 of posts 150 includes a diameter (D4). The diameter (D4) is an inner diameter measured from the inner walls 152 of the posts 150. In embodiments, the diameter (D4) may range between about 0.5 millimeters and about 5 millimeters.

[0055] For example, in embodiments, the diameter (D4) may range between about 0.5 millimeters and about 2.5 millimeters, between about 2.5 millimeters and about 5.0 millimeters, between about 0.5 millimeters and about 1.0 millimeters, between about 1.0 millimeter and about 1.5 millimeter, between about 1.5 millimeters and about 2 millimeters, between about 2 millimeters and about 2.5 millimeters, between about 2.5 millimeters and about 3.0 millimeters, between about 3.0 millimeters and about 3.5 millimeters, between about 3.5 millimeters and about 4.0 millimeters, between about 4.0 millimeters and about 4.5 millimeters, or between about 4.5 millimeters and about 5.0 millimeters, including all ranges and subranges therebetween.

[0056] Each post comprises a height (H4) that may range between about 1 millimeter and about 6 millimeters. For example, in some embodiments, the height (H4) may range between about 1 millimeter and about 1.5 millimeters, between about 1.5 millimeters and about 2 millimeters, or between about 2 millimeters and about 2.5 millimeters, between about 2.5 millimeters and about 3 millimeters, between about 3 millimeters and about 3.5 millimeters, between about 3.5 millimeters and about 4 millimeters, between about 4 millimeters and about 4.5 millimeters, between about 4.5 millimeters and about 5 millimeters, between about 5 millimeters and about 5.5 millimeters, or between about 5.5 millimeters and about 6 millimeters. The height (H4) is measured from the bottom 116 of the well 102. Thus, if the bottom 116 of each well 102 includes a cavity, the height (H4) of the posts 150 may be shorter than if the bottom 116 of each well 102 does not include a cavity. The posts 150 may be arranged to retain a volume of one of the droplets of supporting matrix having suspended cells 180 within the radially spaced apart posts 150 ranging between about 5 microliters and about 50 microliters. A ratio of the height (H4) to the diameter (D4) may range from about 10% to about 30%, for example from about 10% to 12%, from about 10% to about 14%, from about 10% to about 16%, from about 10% to about 18%, from about 10% to about 20%, from about 12% to about 20%, from about 14% to about 20%, from about 16% to about 20%, from about 18% to 20%, from about 20% to about 30%, from about 22% to about 30%, from about 24% to about 30%, from about 26% to about 30%, or from about 28% to about 30%, including all ranges and subranges therebetween. The posts 150 of each retention structure 130 may be configured to retain a volume of one of the assay samples deposited within the retention structure 130 ranging between about 5 microliters and about 50 microliters. However, the supporting matrix may expand in volume due to the absorption of culture media when the culture media is added to the dome culture. Thus, the volume of the assay sample may increase to an expanded volume of 100 microliters or more.

[0057] The raised wall 136 shown in FIGS. 5 and 6, the cavity wall 134 shown in FIG. 7, and the posts 150 shown in FIG. 8 have a relatively short height (H) compared to the diameter (D) of the retention structure 130 to avoid substantial interference with dome recovery, while still (i) providing a physical barrier that protects the organoid dome from being detached due to liquid movement during medium exchange or drug treatment; and (ii) helping to retain the shape and position of the individual organoid domes. The retention structures 130 may comprise an optically transparent material. In embodiments, the retention structures 130 may be optically translucent. In some embodiments, the microplate may also be optically transparent or optically translucent.

[0058] In some embodiments, as shown in FIG. 9, each retention structure 130 may include an inwardly beveled edge 160 along the top portion 140 of the raised wall 136. The beveled edge 160 may have an angle less than about 90 degrees, for example less than about 75 degrees, less than 65 degrees, less than 60 degrees, or less than 50 degrees. Alternatively, the retention structures 130 may include an inwardly chamfered edge along the top portion 140 having an angle less than about 90 degrees, for example less than about 75 degrees, less than 65 degrees, less than 60 degrees, or less than 50 degrees. In other embodiments, the retention structures 130 may include a rounded edge. Fabricating the retention structures 130 with a beveled, chamfered, or rounded edge can reduce the sharpness of the edge of the top portion 140 and improve mechanical retention of the retention structures 130.

[0059] As shown in FIG. 10, the microplate 100 may include a plurality of wells 102 comprising a retention structure 130a positioned on the bottom surface 118 of the well 102 that is in fluid communication with a set of retention structures 130b positioned on the bottom surface 118 of the well 102. In embodiments, the retention structure 130a may be centrally positioned on the bottom surface 118 of the well 102 while the set of retention structures 130b are radially spaced from the retention structure 130a. In embodiments, the retention structure 130a may be in fluid communication with a set of second retention structures 130b through a set of fluidic channels 170 extending between the retention structure 130a and the retention structures 130b to provide fluid communication therebetween. Each fluidic channel 170 comprises an inlet 172 for receiving cells suspended in the supporting matrix from the retention structure 130a and an outlet 174 for delivering cells suspended in the supporting matrix to at least one of the retention structures 130b. Thus, multiple domes can be formed with a single pipetting of a given volume of assay sample comprising cells suspended in a supporting matrix.

[0060] Turning to FIGS. 11A and 11B, the cavities 132 in the microplate 100 in embodiments may be formed by a hot emboss molding method. In such a process, the microplate 100 is manufactured by first softening the microplate bottom wall 112 material and then pressing the material against a mold 190. Thus, the shape of the mold 190 may be pressed and formed into the microplate bottom wall 112. In some embodiments, the mold 190 may be in the shape of a polygonal column configured to form a polygonal cavity in the bottom wall 112. In other embodiments, the shape of the mold 190 may be a cylindrical or elliptic cylinder. In some embodiments, the raised wall 136 may be formed from excess material pushed out of the cavity during the formation of the cavities 132. In some embodiments, the raised wall 136 may be a continuous wall 138 (as shown in FIG. 5) and in other embodiments, the raised wall 136 may be a discontinuous wall 142 (as shown in FIG. 6). After hot embossing the cavities 132, the microplate 100 may be cooled. In some embodiments, the bottom wall of the microplate 100 may have a thickness ranging between about 0.05 millimeters and about 1 millimeters between about 0.05 millimeters and about 0.5 millimeters, between about 0.5 millimeters and about 0.8 millimeters, or between about 0.8 millimeters and about 1.0 millimeters, including all ranges and subranges therebetween. The hot embossed cavities may have a bottom thickness between about 0.02 and about 0.80 millimeters, between about 0.02 millimeters and about 0.1 millimeters, between about 0.1 millimeters and about 0.2 millimeters, or between about 0.2 millimeters and about 0.8 millimeters, including all ranges and subranges therebetween.

[0061] In embodiments, the microplate 100 having cavities 132 may be formed by injection molding. In such a process, a microplate 100 thermoplastic material is liquefied and then injected into a mold cavity of an injection molding machine. The mold cavity comprises shapes forming the microplate 100 comprising a plurality of wells 102 having cavities 132 as shown in FIG. 7. In some embodiments, the shape of the cavities 132 formed may be polygonal. In other embodiments, the shape of the cavities 132 formed may be cylindrical or elliptical. In some embodiments, the mold cavity may further comprise shapes forming a raised wall 136 encompassing each of the cavities 132 (as shown in FIG. 5 and FIG. 1 IB). In some embodiments, the raised wall 136 may be a continuous wall 138 and in other embodiments, the raised wall 136 may be a discontinuous wall 142 (as shown in FIG. 6). Once the thermoplastic material is injected into the mold cavity, the injection molding machine cools the thermoplastic materials to solidify the material and create the microplate 100. The microplate may then be removed. In some embodiments, the bottom wall of the microplate 100 may have a thickness ranging between about 0.05 millimeters and about 1 millimeters, between about 0.05 millimeters and about 0.5 millimeters, between about 0.5 millimeters and about 0.8 millimeters, or between about 0.8 millimeters to about 1.0 millimeters, including all ranges and subranges therebetween. The cavities 132 may comprise a bottom wall thickness ranging between about 0.02 to about 0.80 millimeters, between about 0.02 millimeters and about 0.1 millimeters, between about 0.1 millimeters and about 0.2 millimeters, or between about 0.2 millimeters and about 0.8 millimeters, including all ranges and subranges therebetween.

[0062] In embodiments, the microplate 100 having cavities 132 may be formed by casting. In this process, a microplate 100 thermoplastic material is liquified and then introduced into a mold cavity. The mold cavity comprises shapes forming the microplate 100 comprising a plurality of wells 102 having cavities 132 as shown in FIG. 7. In some embodiments, the shape of the cavities 132 formed may be polygonal. In other embodiments, the shape of the cavities 132 formed may be cylindrical or elliptical. In embodiments, the mold cavity may further comprise shapes forming a raised wall 136 encompassing each of the cavities 132 (as shown in FIGS. 5 and 1 IB). In some embodiments, the raised wall 136 may be a continuous wall 138 and in other embodiments, the raised wall 136 may be a discontinuous wall 142 (as shown in FIG. 6). The liquified thermoplastic material is allowed to solidify to create the microplate 100. The microplate is then removed. The bottom wall of the microplate 100 may have a thickness ranging between about 0.05 millimeters and about 1 millimeters, and the cavities 132 may comprise a bottom thickness ranging between about 0.02 to about 0.80 millimeters. In embodiments, the bottom wall of the microplate 100 may have a thickness ranging between about 0.05 millimeters and about 0.5 millimeters, between about 0.5 millimeters and about 0.8 millimeters, between about 0.8 millimeters and about 1.0 millimeters, between about 0.02 millimeters and about 0.1 millimeters, between about 0.1 millimeters and about 0.2 millimeters, or between about 0.2 millimeters and about 0.8 millimeters, including all ranges and subranges therebetween.

[0063] FIG. 12 presents a summary of the above teachings for using the above-described microplate 100. The flow chart of FIG. 12 illustrates an operation of the method 200. According to step 202, a microplate 100 comprising a bottom wall 112 comprising a plurality of wells 102 arranged in rectilinear arrays of rows and columns is provided. Each well 102 comprises a bottom surface 118 for receiving assay samples 180 of suspended cells in a supporting matrix. The bottom surface 118 comprises a plurality of retention structures 130 to provide mechanical retention of one of the assay samples 180 within each retention structure 130, wherein each retention structure 130 comprises a diameter that may range between about 0.5 millimeters and about 5 millimeters and a height, and wherein a ratio of the height (Hi) to the diameter (Di) may range from about 10% to about 30%.

[0064] For example, the diameter may range between about 0.5 millimeters and about 5.0 millimeters, for example between about 0.5 millimeters and about 1.0 millimeters, between about. 1.0 millimeter and about 1.5 millimeter, between about 1.5 millimeters and about 2 millimeters, between about 2 millimeters and about 2.5 millimeters, between about 2.5 millimeters and about 3.0 millimeters, between 3.0 millimeters and about 3.5 millimeters, between about 3.5 millimeters and about 4.0 millimeters, between about 4.0 millimeters and about 4.5 millimeters, or between about 4.5 millimeters and about 5.0 millimeters, including all ranges and subranges therebetween.

[0065] In some embodiments, the height (Hi) to diameter (Di) ratio of the retention structure 130 may be about 10% to 12%, 10% to 14%, 10% to 16%, or 10% to 18%, 10% to 20%, 12% to 20%, 14% to 20%, 16% to 20%, 18% to 20%, 20% to 30%, 22% to 30%, 24% to 30%, 26% to 30%, or 28% to 30%, including all ranges and subranges therebetween.

[0066] According to optional step 204, the microplate 100 may be warmed before the assay sample 180 is deposited within each of the retention structures 130. The microplate 100 may be warmed in an incubator at a temperature in a range from about 36°C to about 40°C. For example, the microplate 100 may be warmed overnight in an incubator at a temperature of 37° C. An assay sample 180 may be deposited within each of the retention structures 130 according to step 206. The assay samples 180 may then be incubated within each of the retention structures 130 to polymerize the supporting matrix according to step 208. Then, according to step 210, the assay samples may be overlaid with media containing niche factors to form organoids in the supporting matrix retained within each of the retention structures 130. FIG. 12 provides at step 212 that the organoids can be imaged. Three-dimensional imaging or four-dimensional imaging can be used to study the cellular structure of the organoids. Alternatively or additionally, according to step 214, an experiment can be conducted on the organoids. For example, organoid assays can be conducted on the organoids, including but not limited to drug sensitivity assays and growth and viability assays, and other in vivo assays such as RNA and DNA isolation, immunohistochemistry, and genetic manipulation. In one embodiment, the organoids can be propagated and expanded by removing the supporting matrix and dissociating the cells enzymatically or mechanically. The organoids can then be returned to culture conditions.

[0067] Thus, the present disclosure provides a microplate having retention structures, and a method of using the same. The present disclosure contemplates that many changes and modifications may be made. Therefore, while embodiments of the microplate form and methods of using the same have been shown and described, persons skilled in the art will readily appreciate that various additional changes and modifications may be made without departing from the scope of the disclosure.

Claims

What is claimed is:

1. A microplate comprising a plurality of wells for receiving assay samples of cells suspended in supporting matrix, each well of the plurality of wells comprising a bottom surface comprising at least one retention structure to provide mechanical retention of one of the assay samples, the at least one retention structure comprising a diameter ranging between about 0.5 millimeters and about 5 millimeters and a height, and a ratio of the height to the diameter ranges from about 10% to about 30%.

2. The microplate of claim 1, wherein each well of the plurality of wells comprises a plurality of retention structures configured to receive one of the assay samples.

3. The microplate of claim 1, wherein the at least one retention structure comprises a continuous wall, and the height of the at least one retention structure ranges between about 1 millimeter and about 3 millimeters.

4. The microplate of claim 3, wherein the continuous wall is configured to retain a volume of one of the assay samples deposited within the continuous wall ranging between about 5 microliters and about 50 microliters.

5. The microplate of claim 1, wherein the at least one retention structure comprises a shape having a closed perimeter.

6. The microplate of claim 1, wherein the at least one retention structure comprises a plurality of discontinuous walls, and the height of the at least one retention structure ranges between about 1 millimeter and about 3 millimeters.

7. The microplate of claim 6, wherein the discontinuous walls are configured to retain a volume of one of the assay samples deposited within the discontinuous walls ranging between about 5 microliters and about 50 microliters.

8. The microplate of claim 1, wherein the at least one retention structure comprises a plurality of radially spaced apart posts, each post comprising a height ranging between about 1 millimeter and about 3 millimeters.

9. The microplate of claim 8, wherein the plurality of radially spaced apart posts are configured to retain a volume of one of the assay samples deposited within the radially spaced apart posts ranging between about 5 microliters and about 50 microliters.

10. The microplate of claim 1, wherein each well of the plurality of wells includes a first retention structure centrally positioned on the bottom surface and in fluid communication with a set of second retention structures positioned on the bottom surface and spaced from the first retention structure.

11. The microplate of claim 10, further comprising a plurality of fluidic channels extending between the first retention structure and the set of second retention structures to provide fluid communication therebetween, each fluidic channel comprising an inlet for receiving cells suspended in the supporting matrix from the first retention structure and an outlet for delivering cells suspended in the supporting matrix to one of the second retention structures in the set of second retention structures.

12. The microplate of claim 1, wherein the plurality of wells is arranged in rectilinear arrays of rows and columns to form a matrix, and each well of the plurality of wells is a flat bottom well.

13. The microplate of claim 12, wherein the matrix comprises a total of 6 wells, 12 wells, 24 wells, or 48 wells.

14. The microplate of claim 1, wherein the at least one retention structure is formed by injection molding, hot pin embossing, or casting.

15. The microplate of claim 1, wherein the at least one retention structure includes a top edge formed with one of a beveled edge, a rounded edge, or a chamfered edge.

16. The microplate of claim 1, wherein the at least one retention structure is optically transmissive.

17. A method of using a microplate comprising: warming a microplate comprising a plurality of wells for receiving assay samples of suspended cells in a supporting matrix and arranged in rectilinear arrays of rows and columns, each well of the plurality of wells comprising a bottom surface comprising a plurality of retention structures to provide mechanical retention of one of the assay samples within each retention structure, each retention structure comprising a diameter ranging between about 0.5 millimeters and about 5 millimeters and a height, and wherein a ratio of the height to the diameter ranges from about 10% to about 30%; and depositing an assay sample within each of the retention structures.

18. The method of claim 17, further comprising: incubating the assay samples within each of the retention structures to polymerize the supporting matrix; and overlaying the assay samples with media containing niche factors to form organoids in the supporting matrix retained within each of the retention structures.

19. The method of claim 18, further comprising imaging the organoids.

20. The method of claim 17, further comprising warming the microplate before the assay sample is deposited within each of the retention structures.

21. A microplate comprising: a unitary molded microplate comprising (i) a rectangular frame including a planar surface and sidewalls; (ii) a plurality of wells within the planar surface, each well of the plurality of wells comprising a bottom; and (iii) a plurality of optically-transmissive retention structures protruding from the bottom of each well of the plurality of wells, each optically-transmissive retention structure configured to mechanically retain an assay sample of suspended cells in a supporting matrix, each optically-transmissive retention structure comprising a diameter ranging between about 0.5 millimeters and about 5 millimeters and a height, and a ratio of the height to the diameter ranges from about 10% to about 30%.

22. The microplate of claim 21 , wherein the plurality of optically transmissive retention structures was formed by molding recesses in the bottom of each of the plurality of wells.

23. The microplate assay of claim 21, wherein the plurality of optically transmissive retention structures was formed by hot pin embossing recesses into the bottom of each well of the plurality of wells.