Microplates with holding structures and methods of using same

Abstract

1. A microplate comprising a plurality of wells for receiving domes of a support matrix, each well of the plurality of wells comprising a bottom surface having at least one retention structure for providing mechanical retention of one of the assay samples, the at least one retention structure comprising a diameter and a height in the range of about 0.5 millimeters to about 5 millimeters, the ratio of height to diameter being in the range of about 10% to about 30%. 2. A microplate comprising a plurality of wells for receiving domes of a support matrix, each well of the plurality of wells comprising a bottom surface having at least one retention structure for providing mechanical retention of one of the assay samples, the at least one retention structure comprising a diameter and a height in the range of about 0.5 millimeters to about 5 millimeters, the ratio of height to diameter being in the range of about 10% to about 30%.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Application No. 63 / 426,845, filed November 21, 2022, the contents of which are incorporated herein by reference in their entirety.

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

[0003] Although two-dimensional cell culture, which provides a flat monolayer of cells, has helped researchers gain substantial knowledge and understanding of cell behavior, cell function, and cell differentiation, researchers have observed that two-dimensional cell culture provides significantly different behaviors in cell polarity, stem cell differentiation, migration, gene expression, and tissue organization. To create a cellular environment that more closely mimics the in vivo cellular environment, researchers have used three-dimensional scaffolds to cultivate organoids. A support matrix, such as a hydrogel, is used as the three-dimensional scaffold in the organoid culture dome method.

[0004] The present disclosure relates to a technology for a three-dimensional organoid culture dome method. The organoid culture dome method is an established procedure for forming dome structures within the wells of a microplate using a support matrix. Although three-dimensional dome structures are commonly used in organoid models, the organoid culture dome method has several drawbacks. First, forming three-dimensional dome structures within the wells of a microplate requires extensive training and practice by an individual, and even then, results can be variable depending on the individual's skill level. For example, because an individual needs to quickly disperse droplets within the well to avoid gelling of the support matrix during the process, the size of the dome structure can vary significantly. Having dome size variation is not optimal for the consistency of organoid culture. Second, domes are typically placed randomly when multiple domes are positioned within a well. If the domes are placed too close to each other, the proximity of adjacent domes can cause dome fusion. Furthermore, if an individual places the dome too close to the sidewall of the microplate well, the dome may collapse. Yet another common problem with the formation of dome structures is the loss of the dome during the medium exchange process as a result of medium penetration between the dome and the surface of the microplate well. Summary of the Invention

[0005] In view of the above, the above-mentioned challenges may be addressed at least in part by providing at least one retention structure within the wells of the microplate to provide mechanical retention for the domes of the support matrix.

[0006] An exemplary embodiment of the present disclosure provides a microplate comprising a plurality of wells for receiving domes of a support matrix, each well of the plurality of wells comprising a bottom surface comprising at least one retention structure for providing mechanical retention of one of the assay samples, wherein the at least one retention structure may have a diameter and height ranging from about 0.5 millimeters to about 5 millimeters, and the ratio of height to 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 holding structures configured to receive one of the assay samples.

[0008] In some embodiments, the at least one retaining structure may comprise a continuous wall, and the height of the at least one retaining structure may range from about 1 millimeter to 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 from about 5 microliters to about 50 microliters.

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

[0010] In other embodiments, the at least one retaining structure may comprise a plurality of radially spaced posts, each having a height ranging from about 1 millimeter to about 3 millimeters. In some embodiments, the plurality of radially spaced posts may be configured to retain a volume of the assay sample deposited within the radially spaced posts ranging from about 5 microliters to about 50 microliters.

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

[0012] According to some embodiments, the plurality of wells may be arranged in a linear array 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 can range from about 0.05 millimeters to about 1 millimeter, and the at least one retention structure can be formed by injection molding, hot pin embossing, or casting. In some embodiments, the at least one retention structure can include a top edge formed with one of a beveled edge, a rounded edge, or a chamfered edge. In some embodiments, the at least one retention structure can be optically transparent.

[0014] Another exemplary embodiment of the present disclosure provides a method of using a microplate, the method comprising: providing a microplate comprising a plurality of wells arranged in a linear array of rows and columns for receiving assay samples of suspended cells in a support matrix, each well of the plurality of wells comprising a bottom surface comprising a plurality of retention structures for providing mechanical retention of one of the assay samples within each retention structure, each retention structure may have a diameter and height in the range of about 0.5 millimeters to about 5 millimeters, and the ratio of height to diameter may be in the range of about 10% to about 30%; and depositing the assay sample in each of the retention structures.

[0015] The method may, in some embodiments, include incubating a support matrix within each of the holding structures to polymerize the support matrix, and overlaying the support matrix with a medium containing niche factors to form organoids held within the holding structures within each of the support matrices.

[0016] In some embodiments, the method may further include imaging the organoids. In some embodiments, the method may include performing an experiment using the organoids in the support matrix held within each of the holding structures. Further, in some embodiments, the method may include warming the microplate before an assay sample is deposited within each of the holding structures.

[0017] Another exemplary embodiment of the present disclosure comprises a single-piece molded microplate, the single-piece molded microplate comprising: (i) a rectangular frame including a planar surface and sidewalls; (ii) a plurality of wells in the planar surface, each well of the plurality of wells comprising a bottom; and (iii) a plurality of optically transparent retaining structures protruding from the bottom of each of the plurality of wells, each optically transparent retaining structure being configured to mechanically retain an assay sample of suspended cells in a support matrix, each optically transparent retaining structure having a diameter that may range from about 0.5 millimeters to about 5 millimeters, and a height, the ratio of height to diameter being about 10% to about 30%. In some embodiments, the plurality of optically transparent retaining structures are formed by molding a recess in the bottom of each of the plurality of wells. In another embodiment, the plurality of optically transparent retaining structures are formed by hot-pin embossing a recess in the bottom of each of the plurality of wells.

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

[0019] By way of example only, various embodiments are disclosed in reference to the following accompanying schematic drawings, in which corresponding reference symbols indicate corresponding parts: Like reference numbers in different drawing views indicate identical or functionally similar structural elements. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a top view of an exemplary microplate having retaining structures within multiple wells. [Figure 2] FIG. 2 is a partial top view of the exemplary microplate shown in FIG. 1. [Figure 3] 3A is a cross-sectional view of multiple wells of an exemplary microplate taken from line 3-3 of FIG. 3. [Figure 4]4 is a cross-sectional view of multiple wells of an exemplary microplate taken from line 4-4 of FIG. 3. [Figure 5] 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 support matrix. FIG. [Figure 6] FIG. 5 is a cross-sectional view of one of the retention structures shown in FIGS. 3 and 4, except showing the discontinuous walls. [Figure 7] FIG. 5 is a cross-sectional view of one of the retention structures shown in FIGS. 3 and 4, except showing a cavity having a dome of support matrix without a wall protruding from the bottom of the well. [Figure 8] FIG. 1 is a perspective view of an exemplary microplate well having a set of posts forming each of the retention structures. [Figure 9] 5 is a cross-sectional view of the retention structure shown in FIGS. 3 and 4, except without the cavity and the retention structure has beveled edges. FIG. [Figure 10] FIG. 1 is a top view of an exemplary microplate having retention structures and showing channels fluidically connecting the retention structures. [Figure 11A] 11 is a cross-sectional view of one of the retaining structures within one of the wells of the exemplary microplate taken from line 11-11 of FIG. 10, showing hot pin embossing to form a cavity. [Figure 11B] FIG. 11B is a cross-sectional view of the retention structure shown in FIG. 11A after hot pin embossing. [Figure 12] 1 is a flowchart of a method of using a microplate according to an exemplary embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0021] This disclosure is not limited to the particular methodology, materials, and modifications described, as these may, of course, vary. Further, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the claims.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods, devices, or materials similar or equivalent to those described herein may be used in the practice or testing of exemplary embodiments.

[0023] As used herein, the term "substantially" is synonymous with terms such as "near," "very near," "about," "approximately," "approximately," "close," "essentially," "neighborhood," and "vicinity," and such terms may be used interchangeably as they appear in the specification and claims. It should be understood that the term "proximate" is synonymous with terms such as "near," "close," "adjacent," "neighboring," "proximate," and "adjacent," and such terms may be used interchangeably as they appear in the specification and claims. The term "approximately" is intended to mean a value within 10 percent of a specified value. The term "about" and its cognates 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, and measurement error, as well as other factors known to those of skill in the art. When the term "about" is used in describing a value or endpoint of a range, the disclosure should be understood to include the specific value or endpoint referenced. Regardless of whether a numerical value or endpoint of a range in the specification is described as "about," that numerical value or endpoint of the range is intended to include two embodiments: one modified by "about" and one not modified by "about." It will be further understood that each endpoint of a range is significant both in relation to the other endpoint, and independently of the other endpoint.

[0024] The use of "or" in this application refers to a "non-exclusive" arrangement unless otherwise specified. For example, when stating "item x is A or B," it is understood that this can mean one of the following: (1) item x is either A or B, or the other; or (2) item x is both A and B. In other words, the word "or" is not used to define an "exclusive or" arrangement. For example, the "exclusive or" arrangement of the statement "item x is A or B" requires that x can be either A or B, or both. Furthermore, as used herein, "and / or" is intended to refer to a grammatical conjunction used to indicate that one or more of the listed elements or conditions 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 interpreted 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.

[0025] Furthermore, as used herein, the phrases "comprising at least one of" and "comprising at least one of," in connection with a system or element, are 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 interpreted as any one of the following structural arrangements: a device comprising the first element, a device comprising the second element, a device comprising the third element, a device comprising the first element and the second element, a device comprising the first element and the third element, a device comprising the first element, the second element, and the third element, or a device comprising the second element and the third element. A similar interpretation is intended when the phrase "used in at least one of" is used herein.

[0026] References herein to the location of elements (e.g., "top," "bottom," "up," "down," etc.) are used merely to describe the orientation of the various elements in the drawings. The orientation of the various elements may differ according to other exemplary embodiments, and such variations are intended to be encompassed by the present disclosure.

[0027] Specific preferred values ​​disclosed for components, ingredients, additives, dimensions, conditions, times, and similar aspects, and ranges thereof, are for illustrative purposes only and do not exclude other defined values ​​or other values ​​within the defined ranges. The compositions, articles, and methods of the present disclosure can include any value or any combination of the values, specific values, more specific values, and preferred values ​​set forth herein, including any stated or implied intermediate values ​​and ranges.

[0028] The present disclosure describes a microplate with a holding structure and a method for using the same. The holding structure can provide control for positioning organoid domes within the wells of the microplate. Having at least one holding structure within the wells of the microplate can allow for more uniform distribution of organoid domes within the wells, and therefore allow nutrients from the medium to be more evenly used among the domes during culture. This can also help standardize and improve the quality of organoid culture. Furthermore, a microplate with a holding structure can provide better control of the size of organoid domes, especially during manual manipulation by an individual. Furthermore, the holding structure can provide physical support and protection for the organoid domes, reducing the loss of organoid domes during manipulation procedures such as medium changes or drug treatments.

[0029] Turning now to the figures, FIGS. 1-4 show various views of a microplate 100 having a plurality of wells 102. In various embodiments, the microplate 100 may be a single-piece molded microplate having a deck 104 formed of a rectangular frame 106 having a flat surface 108, side walls 110, and a bottom wall 112. Each of the plurality of wells 102 of the microplate 100 further includes a bottom 116 having well walls 114 and a bottom surface 118. In the illustrated example of FIGS. 3 and 4, the plurality of wells 102 may be cylindrical in shape and have a flat bottom 116. The deck 104 may support a linear array of rows and columns to form a matrix, although other array configurations may be employed in further embodiments. 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 with a total of 6 wells, 12 wells, 48 ​​wells, or more. Deck 104 may be formed with other geometric shapes depending on the desired number of wells and / or desired use of microplate 100. Microplate 100 may optionally include a microplate cover (not shown) and a microplate base (not shown).

[0030] The microplate 100 may be made of a polymeric material. In some embodiments, the microplate 100 may comprise polystyrene. For example, the microplate 100 may 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, polyvinyl chloride (PVC), polyethylene terephthalate, a UV-transparent material, a glass material, a 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, which may or may not be treated. In some embodiments, bottom surface 118 may be tissue culture treated or coated with poly-D-lysine (PDL), collagen, fibronectin, a Corning® BioCoat™ surface, or a Corning® PureCoat™ surface. Alternatively, in some embodiments, bottom surface 118 is a Corning® CellBIND® surface.

[0031] Each well 102 may include at least one retention structure 130 for mechanically holding the support matrix, as described below. In one embodiment, organoid cells may be mixed with a support matrix that provides a three-dimensional scaffold. In some embodiments, organoid cells are mixed with the support matrix using the organoid culture dome method. Typically, the support matrix is ​​similar to an extracellular matrix, e.g., a hydrogel, that supports the growth and differentiation of organoids. An exemplary support matrix that may be used is Corning® Matrigel® Matrix. In one exemplary method, the support matrix may be placed on ice to maintain a low temperature of approximately 0°C, and then a droplet of suspended cells ranging from about 5 microliters to about 50 microliters is dispersed within the confines of the retention structure 130. The size of the retention structure 130 may be selected based on the desired size of the organoid dome, as discussed in more detail below. Additionally, other support matrix materials may be used. Similar sized droplets are disposed within wells 102 and adjacent holding structures 130 within other wells 102. The microplate 100 can be preheated in an incubator at about 36°C to about 40°C. In one embodiment, the incubator can be warmed to approximately 37°C before the droplets are dispersed in the microplate 100. In an embodiment, the microplate 100 can be preheated overnight. The size of the droplets can be determined, at least in part, by the number of wells in each well and the height and diameter of the holding structure 130, as discussed in more detail below. Once the cell and support matrix mixture is dispersed on the bottom surface 118 of each well 102, the support matrix can be incubated at 37°C to polymerize the gel and form a dome structure. In some embodiments, the support matrix can be gelled for about 5 to about 10 minutes, whereby the gelled material becomes a semi-solid dome structure with organoid cells embedded in the structure. A medium containing niche factors can be applied to the polymerized domes to allow organoids to form and expand.As used herein, the term "assay sample" refers to a droplet of cells within a support matrix that forms an organoid dome structure.

[0032] In embodiments, each well 102 may include a single retention structure 130. In some embodiments, each well may contain multiple retention structures 130. In certain embodiments, each well 102 may include approximately 1 to 50 retention structures. For example, the wells of a 24-well plate may include 5 to 7 retention structures 130 on the bottom 116 of each well, while a 6-well plate may include 20 to 30 retention structures 130 on the bottom 116 of each well. The retention structures 130 mechanically hold droplets of cells suspended in a scaffolding material similar to the extracellular matrix, also referred to as a support matrix, within the boundaries defined by the retention structures 130, as described in more detail below.

[0033] As shown in FIGS. 3-7 , each retention structure 130 may include a cavity 132 defined by walls 134 and a cavity bottom 128, providing a three-dimensional area for holding a droplet of support matrix with suspended cells 180 forming a dome structure. The size of the retention structure 130 and the number of wells 102 in the microplate 100 may 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 include a raised wall 136 protruding from the bottom surface 118 of the well 102 and the 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 (D1) and a height (H1). The diameter (D1) is the inner diameter measured from the inner wall of the retention structure 130. The height (H1) is measured from the cavity bottom surface 128 to the top surface 140 of the continuous wall 138. In embodiments, the diameter (D1) can range from about 0.5 millimeters to about 5 millimeters. For example, in some embodiments, the diameter (D1) can be in the range of about 0.5 millimeters to 2.5 millimeters, about 2.5 millimeters to 5.0 millimeters, about 0.5 millimeters to 1.0 millimeters, about 1.0 millimeters to about 1.5 millimeters, about 1.5 millimeters to about 2 millimeters, about 2 millimeters to about 2.5 millimeters, about 2.5 millimeters to about 3.0 millimeters, about 3.0 millimeters to about 3.5 millimeters, about 3.5 millimeters to about 4.0 millimeters, about 4.0 millimeters to about 4.5 millimeters, or about 4.5 millimeters to about 5.0 millimeters, including all ranges and subranges therebetween.

[0034] In embodiments, the height (H1) of the continuous wall 138 of the retention structure 130 can range from about 1 millimeter to about 3 millimeters. For example, in some embodiments, the height (H1) can range from about 1 millimeter to about 1.5 millimeters, from about 1.5 millimeters to about 2 millimeters, from about 2 millimeters to about 2.5 millimeters, or from about 2.5 millimeters to about 3 millimeters, including all ranges and subranges therebetween. The height (H1) and diameter (D1) can be selected based on the desired dome size formed from the dispensed droplet of cells suspended in the support matrix 180. That is, the retention structure 130 can be configured to retain a specific droplet size ranging from about 5 microliters to about 50 microliters.

[0035] For example, in some embodiments, each holding structure 130 can hold a droplet size of about 5 microliters. In other embodiments, each holding structure 130 can hold a droplet size of about 10 microliters. In yet other embodiments, each holding structure 130 can hold a droplet size of about 50 microliters. In certain embodiments, the droplet size can be in the range of about 5 microliters to about 10 microliters, about 10 microliters to about 15 microliters, about 15 microliters to about 20 microliters, about 5 microliters to about 25 microliters, about 20 microliters to about 25 microliters, about 25 microliters to about 30 microliters, about 30 microliters to about 35 microliters, about 25 microliters to about 50 microliters, about 35 microliters to about 40 microliters, about 40 microliters to about 45 microliters, or about 45 microliters to about 50 microliters, including all ranges and subranges therebetween.

[0036] The support structure size, as described above, can correspond to the number of wells 102 in each microplate 100. For example, 5 to 7 domes are typically produced in an average volume of about 5 to about 10 microliters and deposited in a 24-well plate. In some embodiments, the ratio of height (H) to diameter (D) can range from about 10% to about 30%. In some embodiments, the ratio of height (H1) to diameter (D1) can be about 10% to 12%, 10% to 14%, 10% to 16%, or 10% to 18%, about 10% to about 20%, about 12% to about 20%, about 14% to about 20%, about 16% to about 20%, about 18% to about 20%, about 20% to about 30%, about 22% to about 30%, about 24% to about 30%, about 26% to about 30%, or about 28% to about 30%, including all ranges and subranges therebetween. In still other embodiments, the ratio of height (H1) to diameter (D1) can be about 30%. As described above, the continuous wall 138 of the retaining structure 130 can be configured to retain a volume of one of the assay samples deposited within the continuous wall 138 ranging from about 5 microliters to about 50 microliters. However, when culture medium is added to the dome culture, the support matrix may expand in volume due to the absorption of the culture medium. 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 about 2 millimeters to about 2.5 millimeters, about 2.5 millimeters to about 3.0 millimeters, about 3.0 millimeters to about 3.5 millimeters, or about 3.5 millimeters to about 4.0 millimeters, including all ranges and subranges therebetween.

[0037] As shown in FIG. 6 , in embodiments, the retention structure 130 can include a raised wall 136, which is a discontinuous wall or set of discontinuous walls 142. FIG. 6 illustrates a three-dimensional region for retaining a droplet of support matrix with suspended cells 180, forming a dome structure, but with the droplet 180 removed to reveal the discontinuous walls 142. The discontinuous walls 142 include openings within the periphery 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 the inner diameter measured from the inner wall of the retention structure 130. The height (H2) is measured from the cavity bottom surface 128 to the top surface 140 of the discontinuous walls 142. In embodiments, diameter (D2) can be in the range of about 0.5 millimeters to about 5 millimeters. For example, in embodiments, diameter (D2) can be in the range of about 0.5 millimeters to about 2.5 millimeters, about 2.5 millimeters to about 5.0 millimeters, about 0.5 millimeters to about 1.0 millimeters, about 1.0 millimeters to about 1.5 millimeters, about 1.5 millimeters to about 2 millimeters, about 2 millimeters to about 2.5 millimeters, about 2.5 millimeters to about 3.0 millimeters, about 3.0 millimeters to about 3.5 millimeters, about 3.5 millimeters to about 4.0 millimeters, about 4.0 millimeters to about 4.5 millimeters, or about 4.5 millimeters to about 5.0 millimeters, including all ranges and subranges therebetween.

[0038] In embodiments, the height of discontinuous walls 142 of retention structure 130 can range from about 1 millimeter to about 3 millimeters. For example, in some embodiments, height (H2) can range from about 1 millimeter to about 1.5 millimeters, from about 1.5 millimeters to about 2 millimeters, from about 2 millimeters to about 2.5 millimeters, or from about 2.5 millimeters to about 3 millimeters, including all ranges and subranges therebetween. Height (H2) and diameter (D2) can be selected based on the desired dome size formed from the dispensed droplet of cells suspended in the support matrix. The ratio of height (H2) to diameter (D2) can be in the range of about 10% to about 30%, e.g., about 10% to about 12%, about 10% to about 14%, about 10% to about 16%, about 10% to about 18%, about 10% to about 20%, about 12% to about 20%, about 14% to about 20%, about 16% to about 20%, about 18% to about 20%, about 20% to about 30%, about 22% to about 30%, about 24% to about 30%, about 26% to about 30%, or about 28% to about 30%, including all ranges and subranges therebetween. The discontinuous walls 142 of the retention structure 130 can be configured to retain a volume of one of the assay samples deposited within the discontinuous walls 142 in the range of about 5 microliters to about 50 microliters. However, when culture medium is added to the dome culture, the support matrix can expand in volume due to the absorption of culture medium, and thus the volume of the organoid dome can increase to an expanded volume of 100 microliters or more.

[0039] In alternative embodiments, as illustrated in FIG. 7 , retention structure 130 may include a cavity 132 without raised walls. In such embodiments, cavity 132 defined by cavity walls 134 and cavity bottom 128 provides a three-dimensional area for retaining a droplet of support matrix with suspended cells 180 forming a dome structure. In such embodiments, retention structure 130 includes a diameter (D3) and a height (H3). In embodiments, diameter (D3) is the inner diameter measured from the inner wall of cavity 132. Height (H3) is measured from cavity bottom 128 to the top of cavity 132, which is bottom 118 of well 102. In embodiments, diameter (D3) may range from about 0.5 millimeters to about 5 millimeters.

[0040] For example, in some embodiments, the diameter (D3) can be in the range of about 0.5 millimeters to about 2.5 millimeters, about 2.5 millimeters to about 5.0 millimeters, about 0.5 millimeters to about 1.0 millimeters, about 1.0 millimeters to about 1.5 millimeters, about 1.5 millimeters to about 2 millimeters, about 2 millimeters to about 2.5 millimeters, about 2.5 millimeters to about 3.0 millimeters, about 3.0 millimeters to about 3.5 millimeters, about 3.5 millimeters to about 4.0 millimeters, about 4.0 millimeters to about 4.5 millimeters, or about 4.5 millimeters to about 5.0 millimeters, including all ranges and subranges therebetween.

[0041] In embodiments, the height of cavity wall 134 of retention structure 130 can range from about 0.5 millimeters to about 3 millimeters. For example, in embodiments, height (H3) can range from about 1 millimeter to about 1.5 millimeters, about 1.5 millimeters to about 2 millimeters, about 2 millimeters to about 2.5 millimeters, and about 2.5 millimeters to about 3 millimeters, including all ranges and subranges therebetween. Height (H3) and diameter (D3) can be selected based on the desired dome size formed from the dispensed droplet of cells suspended in the support matrix. The ratio of height (H3) to diameter (D3) can range from about 5% to about 30%. Cavity 132 of retention structure 130 can be configured to hold a single volume of a droplet of support matrix having suspended cells 180 deposited within cavity 132 to form a dome structure, and the volume can range from about 5 microliters to about 50 microliters. In certain embodiments, the volume range can be about 5 microliters to about 10 microliters, about 10 microliters to about 15 microliters, about 15 microliters to about 20 microliters, about 5 microliters to about 25 microliters, about 20 microliters to about 25 microliters, about 25 microliters to about 30 microliters, about 30 microliters to about 35 microliters, about 25 microliters to about 50 microliters, about 35 microliters to about 40 microliters, about 40 microliters to about 45 microliters, or about 45 microliters to about 50 microliters, including all ranges and subranges therebetween. However, it should be understood that the support matrix may expand in volume due to absorption of culture medium. Thus, the volume of the organoid dome can increase to an expanded volume of 100 microliters or more. For example, the organoid dome may increase to an expanded volume of about 10 microliters to about 200 microliters, about 10 microliters to about 50 microliters, about 50 microliters to about 100 microliters, about 100 microliters to about 150 microliters, or about 150 microliters to about 200 microliters, including all ranges and subranges therebetween.

[0042] In other embodiments, as shown in FIG. 8 , the retention structure 130 may include a plurality of radially spaced posts 150, each of which forms a set. It should be understood that each well 102 may include a plurality of retention structures 130. For example, FIG. 8 shows seven retention structures 130, each of which includes a plurality of radially spaced posts 150. Each set of posts 150 forming a retention structure 130 may alternatively form an oval, polygonal, or other shape. In embodiments, the posts 150 may each be cylindrical. However, other shapes are possible, including, but not limited to, an oval or polygonal shape, or a triangle, square, or rectangle. In embodiments, the posts 150 of each retention structure 130 may surround a cavity in the bottom 116 of the well 102, such as cavity 132 as shown in FIG. 7 . In other embodiments, the posts 150 may be disposed on the bottom 116 of the well 102 without a cavity. The retention structure 130 of the posts 150 may include three to four posts. However, additional posts 150 may be used to form the retention structure 130. Each retention structure 130 of the posts 150 includes a diameter (D4). The diameter (D4) is the inner diameter measured from the inner wall 152 of the post 150. In embodiments, the diameter (D4) may range from about 0.5 millimeters to about 5 millimeters.

[0043] For example, in embodiments, the diameter (D4) can be in the range of about 0.5 mm to about 2.5 mm, about 2.5 mm to about 5.0 mm, about 0.5 mm to about 1.0 mm, about 1.0 mm to about 1.5 mm, about 1.5 mm to about 2 mm, about 2 mm to about 2.5 mm, about 2.5 mm to about 3.0 mm, about 3.0 mm to about 3.5 mm, about 3.5 mm to about 4.0 mm, about 4.0 mm to about 4.5 mm, or about 4.5 mm to about 5.0 mm, including all ranges and subranges therebetween.

[0044] Each pillar has a height (H4) that can range from about 1 millimeter to about 6 millimeters. For example, in some embodiments, the height (H4) can range from about 1 millimeter to about 1.5 millimeters, about 1.5 millimeters to about 2 millimeters, or about 2 millimeters to about 2.5 millimeters, about 2.5 millimeters to about 3 millimeters, about 3 millimeters to about 3.5 millimeters, about 3.5 millimeters to about 4 millimeters, about 4 millimeters to about 4.5 millimeters, about 4.5 millimeters to about 5 millimeters, about 5 millimeters to about 5.5 millimeters, or about 5.5 millimeters to about 6 millimeters. The height (H4) is measured from the bottom 116 of the well 102. Thus, when the bottom 116 of each well 102 includes a cavity, the height (H4) of the pillar 150 may be shorter than when the bottom 116 of each well 102 does not include a cavity. The pillars 150 can be arranged to hold a single volume of a droplet of support matrix having suspended cells 180 within the radially spaced pillars 150 ranging from about 5 microliters to about 50 microliters. The ratio of height (H4) to diameter (D4) can be in the range of about 10% to about 30%, e.g., about 10% to about 12%, about 10% to about 14%, about 10% to about 16%, about 10% to about 18%, about 10% to about 20%, about 12% to about 20%, about 14% to about 20%, about 16% to about 20%, about 18% to about 20%, about 20% to about 30%, about 22% to about 30%, about 24% to about 30%, about 26% to about 30%, or about 28% to about 30%, including all ranges and subranges therebetween. The pillars 150 of each holding structure 130 can be configured to hold a volume of one of the assay samples deposited within the holding structure 130 ranging from about 5 microliters to about 50 microliters. However, the support matrix can expand in volume when culture medium is added to the dome culture due to absorption of the culture medium. Thus, the volume of the assay sample can increase to an expanded volume of 100 microliters or more.

[0045] The raised walls 136 shown in Figures 5 and 6, the cavity walls 134 shown in Figure 7, and the posts 150 shown in Figure 8 have a relatively short height (H) compared to the diameter (D) of the holding structure 130 to avoid substantial interference with dome retrieval, yet (i) provide a physical barrier to protect the organoid domes from detachment due to fluid movement during medium exchange or drug treatment, and (ii) help maintain the shape and position of individual organoid domes. The holding structure 130 may comprise an optically transparent material. In embodiments, the holding structure 130 may be optically translucent. In some embodiments, the microplate may be optically transparent or optically translucent.

[0046] 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 of less than about 90 degrees, e.g., less than about 75 degrees, 65 degrees, 60 degrees, or 50 degrees. Alternatively, the retention structure 130 may include an inwardly chamfered edge along the top portion 140, where the chamfered edge has an angle of less than about 90 degrees, e.g., less than about 75 degrees, 65 degrees, 60 degrees, or 50 degrees. In other embodiments, the retention structure 130 may include a rounded edge. Fabricating the retention structure 130 with a beveled, chamfered, or rounded edge may reduce the sharpness of the edge of the top portion 140 and improve the mechanical retention of the retention structure 130.

[0047] As shown in FIG. 10 , a microplate 100 may include a plurality of wells 102 with retaining structures 130a positioned on the bottom surface 118 of the wells 102, the retaining structures 130a being in fluid communication with a set of retaining structures 130b positioned on the bottom surface 118 of the wells 102. In an embodiment, the retaining structure 130a may be positioned in the center of the bottom surface 118 of the wells 102, while the set of retaining structures 130b is radially spaced from the retaining structure 130a. In an embodiment, the retaining structure 130a may be in fluid communication with a second set of retaining structures 130b through a set of fluid channels 170 extending between the retaining structures 130a and 130b to provide fluid communication therebetween. Each fluid channel 170 includes an inlet 172 for receiving cells suspended in a support matrix from the retaining structure 130a and an outlet 174 for delivering the cells suspended in the support matrix to at least one of the retaining structures 130b. Thus, multiple domes can be formed with a single pipetting of a given volume of assay sample containing cells suspended in a support matrix.

[0048] 11A and 11B, the cavities 132 in the microplate 100 of embodiments may be formed by a hot embossing process. In such a process, the microplate 100 is fabricated by first softening the microplate bottom wall 112 material and then pressing the material into a mold 190. Thus, the shape of the mold 190 may be pressed 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 elliptical cylinder. In some embodiments, the raised walls 136 may be formed from excess material extruded from the cavities 132 during their formation. In some embodiments, the raised walls 136 may be continuous walls 138 (as shown in FIG. 5), while in other embodiments, the raised walls 136 may be discontinuous walls 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 from about 0.05 millimeters to about 1 millimeter, from about 0.05 millimeters to about 0.5 millimeters, from about 0.5 millimeters to about 0.8 millimeters, or from about 0.8 millimeters to about 1.0 millimeters, including all ranges and subranges therebetween. The hot embossed cavities may have a bottom thickness ranging from about 0.02 millimeters to about 0.80 millimeters, from about 0.02 millimeters to about 0.1 millimeters, from about 0.1 millimeters to about 0.2 millimeters, or from about 0.2 millimeters to about 0.8 millimeters, including all ranges and subranges therebetween.

[0049] In embodiments, the microplate 100 having cavities 132 may be formed by injection molding. In such a process, the thermoplastic material of the microplate 100 is liquefied and then injected into a mold cavity of an injection molding machine. The mold cavity has a shape that forms the microplate 100 with a plurality of wells 102 having cavities 132, as shown in FIG. 7. In some embodiments, the shape of the formed cavities 132 may be polygonal. In other embodiments, the shape of the formed cavities 132 may be cylindrical or elliptical. In some embodiments, the mold cavity may further have a shape that forms raised walls 136 surrounding each of the cavities 132 (as shown in FIGS. 5 and 11B). In some embodiments, the raised walls 136 may be continuous walls 138, and in other embodiments, the raised walls 136 may be discontinuous walls 142 (as shown in FIG. 6). Once the thermoplastic material is injected into the mold cavity, the injection molding machine cools the thermoplastic material, solidifying the material and creating the microplate 100. The microplate may then be removed. In some embodiments, the bottom wall of the microplate 100 may have a thickness ranging from about 0.05 millimeters to about 1 millimeter, from about 0.05 millimeters to about 0.5 millimeters, from about 0.5 millimeters to about 0.8 millimeters, or from about 0.8 millimeters to about 1.0 millimeters, including all ranges and subranges therebetween. The cavity 132 may have a bottom wall thickness ranging from about 0.02 millimeters to about 0.80 millimeters, from about 0.02 millimeters to about 0.1 millimeters, from about 0.1 millimeters to about 0.2 millimeters, or from about 0.2 millimeters to about 0.8 millimeters, including all ranges and subranges therebetween.

[0050] In embodiments, the microplate 100 having cavities 132 may be formed by casting. In this process, the thermoplastic material of the microplate 100 is liquefied and then introduced into a mold cavity. The mold cavity has a shape that forms the microplate 100 with a plurality of wells 102 having cavities 132, as shown in FIG. 7. In some embodiments, the formed cavities 132 may have a polygonal shape. In other embodiments, the formed cavities 132 may have a cylindrical or elliptical shape. In embodiments, the mold cavity may further have a shape that forms raised walls 136 surrounding each of the cavities 132 (as shown in FIGS. 5 and 11B). In some embodiments, the raised walls 136 may be continuous walls 138, and in other embodiments, the raised walls 136 may be discontinuous walls 142 (as shown in FIG. 6). The liquefied thermoplastic material solidifies to create the microplate 100. The microplate is then removed. The bottom wall of microplate 100 may have a thickness ranging from about 0.05 millimeters to about 1 millimeter, and cavity 132 may have a bottom thickness ranging from about 0.02 to about 0.80 millimeters. In embodiments, the bottom wall of microplate 100 may have a thickness ranging from about 0.05 millimeters to about 0.5 millimeters, from about 0.5 millimeters to about 0.8 millimeters, from about 0.8 millimeters to about 1.0 millimeters, from about 0.02 millimeters to about 0.1 millimeters, from about 0.1 millimeters to about 0.2 millimeters, or from about 0.2 millimeters to about 0.8 millimeters, including all ranges and subranges therebetween.

[0051] FIG. 12 shows a summary of the above teachings for using the microplate 100 described above. The flowchart in FIG. 12 illustrates the operations of a method 200. According to step 202, a microplate 100 is provided having a bottom wall 112 with a plurality of wells 102 arranged in a linear array of rows and columns. Each well 102 has a bottom surface 118 for receiving an assay sample 180 of suspended cells in a support matrix. The bottom surface 118 includes a plurality of retention structures 130 for providing mechanical retention of one of the assay samples 180 within each retention structure 130, each retention structure 130 having a diameter and height that can range from about 0.5 millimeters to about 5 millimeters, and the ratio of height (H1) to diameter (D1) can range from about 10% to about 30%.

[0052] For example, the diameter can range from about 0.5 millimeters to about 5.0 millimeters, e.g., from about 0.5 millimeters to about 1.0 millimeters, about 1.0 millimeters to about 1.5 millimeters, about 1.5 millimeters to about 2 millimeters, about 2 millimeters to about 2.5 millimeters, about 2.5 millimeters to about 3.0 millimeters, 3.0 millimeters to about 3.5 millimeters, about 3.5 millimeters to about 4.0 millimeters, about 4.0 millimeters to about 4.5 millimeters, or about 4.5 millimeters to about 5.0 millimeters, including all ranges and subranges therebetween.

[0053] In some embodiments, the ratio of height (H1) to diameter (D1) of retention structure 130 may be approximately 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.

[0054] According to optional step 204, the microplate 100 may be warmed before the assay sample 180 is deposited into each of the holding structures 130. The microplate 100 may be warmed in an incubator at a temperature ranging from about 36°C to about 40°C. For example, the microplate 100 may be warmed overnight at 37°C in an incubator. The assay sample 180 may be deposited into each of the holding structures 130 according to step 206. The assay sample 180 may then be incubated in each of the holding structures 130 to polymerize the support matrix according to step 208. Then, according to step 210, the assay sample may be overlaid with a medium containing niche factors to form organoids in the support matrix held within each of the holding structures 130. FIG. 12 provides that in step 212, the organoids may be imaged. Three-dimensional or four-dimensional imaging may be used to study the cellular structure of the organoids. Alternatively or additionally, experiments can be carried out on organoids according to step 214. For example, organoid assays can be carried out on organoids, including but not limited to drug sensitivity assays and growth and viability assays, as well as other in vivo assays such as RNA and DNA isolation, immunohistochemistry, and genetic manipulation.In one embodiment, organoids can be grown and expanded by removing support matrix and enzymatically or mechanically dissociating cells.Then, organoids can be returned to culture conditions.

[0055] Thus, the present disclosure provides microplates having holding structures and methods of using the same. The present disclosure contemplates that many variations and modifications may be made. Thus, while an embodiment of a microplate configuration and methods of using the same have been shown and described, those skilled in the art will readily appreciate that various additional variations and modifications may be made without departing from the scope of the present disclosure.

Claims

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

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

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

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

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

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

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

8. 10. The microplate of claim 1, wherein the at least one retention structure comprises a plurality of radially spaced posts, each post having a height ranging from about 1 millimeter to about 3 millimeters.

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

10. 2. The microplate of claim 1, wherein each well of the plurality of wells includes a first retaining structure positioned at a center of the bottom surface and in fluid communication with a set of second retaining structures positioned at the bottom surface and spaced apart from the first retaining structures.

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

12. 2. The microplate of claim 1, wherein the plurality of wells are arranged in a linear array of rows and columns to form a matrix, and each well of the plurality of wells is a flat-bottom well.

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

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

15. 10. The microplate of claim 1, wherein the at least one retaining structure comprises 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 retaining structure is optically transparent.

17. A method using a microplate, comprising: warming a microplate comprising a plurality of wells for receiving assay samples of suspended cells in a support matrix, the plurality of wells being arranged in a linear array of rows and columns, each well of the plurality of wells comprising a bottom surface comprising a plurality of retention structures for providing mechanical retention of one of the assay samples within each retention structure, each retention structure comprising a diameter ranging from about 0.5 millimeters to about 5 millimeters, and a height, the ratio of the height to the diameter ranging from about 10% to about 30%; depositing an assay sample within each of said holding structures.

18. incubating the assay sample within each of the retention structures to polymerize the support matrix; 18. The method of claim 17, further comprising overlaying the assay sample with a medium containing niche factors to form organoids in the support matrix held within each of the holding structures.

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

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

21. A microplate, 1. A 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 light-transmitting retaining structures protruding from the bottom of each well of the plurality of wells, each light-transmitting retaining structure configured to mechanically retain an assay sample of suspended cells in a support matrix, each light-transmitting retaining structure having a diameter ranging from about 0.5 millimeters to about 5 millimeters, and a height, wherein the ratio of the height to the diameter ranges from about 10% to about 30%.

22. 22. The microplate according to claim 21, wherein the plurality of optically transparent holding structures are formed by molding a recess in the bottom of each of the plurality of wells.

23. 22. The microplate assay of claim 21, wherein the plurality of optically transparent holding structures are formed by hot-pin embossing a recess in the bottom of each well of the plurality of wells.

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