Microplates with retention structures and methods of use thereof

Abstract

A microplate comprising a plurality of pores for receiving a dome of a support matrix, each pore of the plurality of pores comprising a bottom surface having at least one retention structure to provide mechanical retention for one of the analytic samples, the at least one retention structure comprises a diameter ranging between about 0.5 mm and about 5 mm and a height, and wherein the ratio of the height to the diameter ranges from about 10% to about 30%; and a method of using the microplate.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority under the Patent Law to U.S. Provisional Application Serial No. 63 / 426,845, filed on November 21, 2022, the content of which is incorporated herein by reference in its entirety and made a part hereof. Technical Field

[0003] This disclosure relates to microplates. More particularly, this disclosure relates to microplates having at least one retention structure within a well to provide mechanical retention for a dome of a support matrix having suspended cells. Background Art

[0004] Although two - dimensional cell culture, which provides a flat monolayer of cells, has helped researchers gain a great deal of knowledge and understanding about cell behavior, cell function, and cell differentiation, researchers have observed that two - dimensional cell culture exhibits significantly different behavior in terms of 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 cultivate organoids. In the organoid culture dome method, a support matrix (such as a hydrogel) is used as the three - dimensional scaffold.

[0005] This disclosure relates to the technology of the three - dimensional organoid culture dome method. The organoid culture dome method is an established procedure for forming a dome structure in 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 a three - dimensional dome structure within the wells of a microplate requires a great deal of training and practice by an individual, and even then, the results may vary depending on the individual's skill level. For example, the size of the dome structure can vary widely because during this process, the individual needs to quickly disperse droplets into the well to avoid gelation of the support matrix. Variations in dome size are not suitable for organoid culture consistency. Second, when multiple domes are placed within a well, the domes are typically randomly positioned. If the domes are placed too close to each other, dome fusion may occur due to the proximity of adjacent domes. Additionally, if an individual places a dome too close to the sidewall of the microplate well, the dome may collapse. Another common problem during the dome structure formation process is that during the medium exchange process, the medium leaks out between the dome and the surface of the microplate well, resulting in dome loss. Summary of the Invention

[0006] In view of the foregoing, the foregoing challenges can be at least partially addressed by providing at least one retention structure within the wells of a microplate to provide mechanical retention for a dome of a support matrix.

[0007] Exemplary embodiments of the present disclosure provide a microplate that includes a plurality of wells for receiving domes of a support matrix. Each well of the plurality of wells includes a bottom surface that includes at least one retention structure to provide mechanical retention for one of the assay samples. The at least one retention structure may include a diameter ranging between about 0.5 millimeters and about 5 millimeters and a height, and the ratio of the height to the diameter may range between about 10% and about 30%.

[0008] In some embodiments, each well of the plurality of wells may include a plurality of retention structures configured to receive one of the assay samples.

[0009] In some embodiments, the at least one retention structure may include a continuous wall, and the height of the at least one retention structure may range between about 1 millimeter and about 3 millimeters. Additionally, in some embodiments, the continuous wall may be configured to retain one of the assay samples having a volume ranging between about 5 microliters and about 50 microliters deposited within the continuous wall.

[0010] In other embodiments, the at least one retention structure may include 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. Additionally, in some embodiments, the discontinuous walls may be configured to retain one of the assay samples having a volume ranging between about 5 microliters and about 50 microliters deposited within the discontinuous walls.

[0011] In other embodiments, the at least one retention structure may include a plurality of radially spaced-apart struts. Each strut may include a height ranging between about 1 millimeter and about 3 millimeters. In some embodiments, the plurality of radially spaced-apart struts may be configured to retain one of the assay samples having a volume ranging between about 5 microliters and about 50 microliters deposited within the radially spaced-apart struts.

[0012] In some embodiments, each well of the plurality of wells may include a first retention structure centrally disposed on the bottom surface and in fluid communication with a set of second retention structures disposed on the bottom surface and spaced apart from the first retention structure. Each well may include a plurality of fluid channels extending between the first retention structure and the set of second retention structures to provide fluid communication therebetween. Each of the fluid channels includes an inlet for receiving cells suspended in the support matrix from the first retention structure and an outlet for delivering the cells suspended in the support matrix to one of the second retention structures of the set of second retention structures.

[0013] According to some embodiments, the plurality of holes may be arranged in a linear array of rows and columns to form a matrix, and each of the plurality of holes may be a flat-bottomed hole. The matrix may include, for example, a total of 6 holes, 12 holes, 24 holes, or 48 holes.

[0014] In some embodiments, the thickness of the bottom wall of the microplate may range between about 0.05 millimeters and about 1 millimeter, and the at least one holding structure may be formed by injection molding, hot pin embossing, or casting. In some embodiments, the at least one holding structure may 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 holding structure may be optically transmissive.

[0015] Another exemplary embodiment of the present disclosure provides a method of using a microplate, comprising: providing a microplate comprising a plurality of holes arranged in a linear array of rows and columns for receiving an assay sample of suspended cells in a support matrix, each of the plurality of holes comprising a bottom surface comprising a plurality of holding structures for providing mechanical retention for one of the assay samples within each holding structure, wherein each holding structure may comprise a diameter ranging between about 0.5 millimeters and about 5 millimeters and a height, and wherein the ratio of the height to the diameter may range from about 10% to about 30%; and depositing the assay sample within each of the holding structures.

[0016] In some embodiments, the method may comprise culturing the support matrix within each of the holding structures to polymerize the support matrix, and overlaying the support matrix with a culture medium containing a niche factor to form organoids in the support matrix retained within each of the holding structures.

[0017] In some embodiments, the method may further comprise imaging the organoids. In some embodiments, the method may include performing experiments using the organoids in the support matrix retained within each of the holding structures. Additionally, in some embodiments, the method may comprise the step of heating the microplate before depositing the assay sample within each of the holding structures.

[0018] Another exemplary embodiment of the present disclosure includes a unitary molded microplate comprising: (i) a rectangular frame including a planar surface and sidewalls; (ii) a plurality of holes within the planar surface, each of the plurality of holes including a bottom; and (iii) a plurality of optically transmissive holding structures protruding from the bottom of each of the plurality of holes, each optically transmissive holding structure being configured to mechanically hold an assay sample of suspended cells in a support matrix, wherein each optically transmissive holding structure includes a diameter ranging from about 0.5 millimeters to about 5 millimeters and a height, and wherein the ratio of the height to the diameter ranges from about 10% to about 30%. In some embodiments, the plurality of optically transmissive holding structures are formed by molding grooves in the bottom of each of the plurality of holes. In another embodiment, the plurality of optically transmissive holding structures are formed by thermally embossing grooves into the bottom of each of the plurality of holes.

[0019] Embodiments of the present disclosure will be described below. However, the present disclosure is not limited to the described embodiments, and various modifications are possible without departing from the basic principles described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Various embodiments are disclosed by way of example only with reference to the following accompanying schematic drawings, in which corresponding reference numerals indicate corresponding parts. Identical reference numerals on different drawing views identify the same or functionally similar structural elements.

[0021] Figure 1 Top plan view of an exemplary microplate having holding structures within a plurality of holes;

[0022] Figure 2 For Figure 1 Partial top plan view of the exemplary microplate shown in

[0023] Figure 3 For Figure 3 Cross-sectional view of a plurality of holes of the exemplary microplate taken along line 3-3 of

[0024] Figure 4 For Figure 3 Cross-sectional view of a plurality of holes of the exemplary microplate taken along line 4-4 of

[0025] Figure 5 For Figure 3 And 4 Cross-sectional view of one of the holding structures shown in

[0026] Figure 6 ForFigure 3 and 4 A cross-sectional view of one of the retention structures shown in 4 , except that a discontinuous wall is shown;

[0027] Figure 7 is Figure 3 and 4 A cross-sectional view of one of the retention structures shown in 4 , except that a cavity without a wall protruding from the bottom surface of the hole is shown and has a dome supporting the substrate;

[0028] Figure 8 Is a perspective view of a hole of an exemplary microplate having groups of struts forming each of the retention structures;

[0029] Figure 9 is Figure 3 and 4 A cross-sectional view of the retention structure shown in 4 , except that it does not have a cavity and the retention structure has a chamfered edge;

[0030] Figure 10 Is a top plan view of an exemplary microplate having a retention structure and showing a channel in fluid communication with the retention structure;

[0031] Figure 11A is from Figure 10 A cross-sectional view of one of the retention structures within one of the holes of an exemplary microplate taken along line 11-11 of Figure 10 , which shows hot embossing forming a cavity;

[0032] Figure 11B is Figure 11A A cross-sectional view of the retention structure shown in Figure 11A after hot embossing; and

[0033] Figure 12 Is a flow chart of a method of using a microplate according to an exemplary embodiment of the present disclosure. Detailed Description

[0034] The present disclosure is not limited to the specific methods, materials, and modifications described, and thus may of course vary. Additionally, the terms used herein are for the purpose of describing particular aspects only and are not intended to limit the scope of the claims.

[0035] 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 practicing or testing the exemplary embodiments.

[0036] 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", and these terms are interchangeable as they appear in the specification and claims. It should be understood that the term "proximate" is synonymous with terms such as "nearby", "close", "adjacent", "neighboring", "immediate", "adjoining", and these terms are interchangeable as they appear in the specification and claims. The term "substantially" is intended to mean a value within ten percent of the specified value. The term "about" and its synonymous terms mean that the quantity, size, formulation, parameter, and other quantities and characteristics are not exact and need not be exact, but may be approximate and / or larger or smaller as needed, thereby reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those skilled in the art. When the term "about" is used to describe a value or endpoint of a range, the present disclosure should be understood to include the recited specific value or endpoint. Whether or not the numerical value or range endpoint in the specification is recited with "about", the numerical value or range endpoint is intended to include two embodiments: one modified by "about", and one not modified by "about". It should be further understood that each endpoint of a range is significant relative to and independent of the other endpoint.

[0037] Unless otherwise stated, in this application, the use of "or" is with respect to a "non-exclusive" arrangement. For example, when stating "Item x is A or B", it should be understood that this can mean one of the following cases: (1) Item x is only one or the other of A and B; (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, 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. Further, as used herein, "and / or" is intended to mean a grammatical conjunction used to indicate that one or more of the recited 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 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 and second elements; a device comprising the first and third elements; a device comprising the first, second, and third elements; or a device comprising the second and third elements.

[0038] Further, as used herein, the phrases "comprises 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 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 and second elements; a device comprising the first and third elements; a device comprising the first, second, and third elements; or a device comprising the second and third elements. When the phrase "for at least one of the following:" is used herein, it is intended to be interpreted in a similar manner.

[0039] References herein to the position of elements (e.g., "top", "bottom", "above", "below", etc.) are only for describing the orientation of the respective elements in the drawings. Depending on other exemplary embodiments, the orientation of the respective elements may be different, and such variations are intended to be covered by this disclosure.

[0040] The specific and preferred values and ranges thereof disclosed with respect to components, ingredients, additives, dimensions, conditions, times, etc. are for illustration only; they do not exclude other defined values or other values within the defined ranges. The compositions, articles, and methods of this disclosure may include any value described herein, or any combination of values, specific values, more specific values, and preferred values, including intermediate values and ranges, whether explicitly or implicitly.

[0041] In the present disclosure, a microwell plate having a retention structure and a method of using the same are described. The retention structure can provide control over the placement of organoid domes within the wells of the microwell plate. Having at least one retention structure within the wells of the microwell plate can allow for a more uniform distribution of organoid domes within the wells and, thus, can allow for a more uniform utilization of nutrients from the culture medium between the domes during culturing. This can also help to standardize and improve the quality of organoid culture. Additionally, a microwell plate having a retention structure can better control the size of organoid domes, especially in the case of individual manual manipulation. Further, the retention structure can provide physical support and protection for the organoid domes and reduce loss of the organoid domes during procedural operations such as media exchange or drug treatment.

[0042] Turning now to the drawings, Figures 1 to 4 various views of a microwell plate 100 having a plurality of wells 102 are shown. In various embodiments, the microwell plate 100 can be a one-piece molded microwell plate having a deck 104 formed by a rectangular frame 106 including a planar surface 108, sidewalls 110, and a bottom wall 112. Each of the plurality of wells 102 of the microwell plate 100 further includes a well wall 114 and a bottom 116 having a bottom surface 118. In Figure 3 and 4 the illustrated example, the plurality of wells 102 can be cylindrical with a flat bottom 116. The deck 104 can support a linear array of rows and columns to form a matrix, but in other embodiments, other array configurations can be employed. For example, as Figure 1 shown in, the deck 104 can support 24 wells. However, the deck 104 can support fewer or additional wells. For example, the microwell plate 100 can include a matrix that in total includes 6 wells, 12 wells, 48 wells, or more wells. Depending on the desired number of wells and / or desired use of the microwell plate 100, the deck 104 can be formed in other geometries. The microwell plate 100 can optionally include a microwell plate lid (not shown) and a microwell plate base (not shown).

[0043] The microplate 100 can be made of a polymeric material. In some embodiments, the microplate 100 can 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 can comprise an optically transparent bottom and a black opaque polystyrene microplate body or a white opaque polystyrene microplate body. The microplate 100 can alternatively be made of polycarbonate, polypropylene, polyvinyl chloride (PVC), polyethylene terephthalate, a UV transparent material, a glass material, a cycloolefin copolymer (COC), or a combination thereof. However, the microplate 100 can be made of other materials. The bottom 116 of each well 102 comprises a bottom surface 118 that can be treated or untreated. In some embodiments, the bottom surface 118 can be tissue culture treated or coated with poly-D-lysine (PDL), collagen, fibronectin, BioCoat TM surface or PureCoat TM surface. Alternatively, in some embodiments, the bottom surface 118 is surface.

[0044] As described below, each well 102 can comprise at least one retention structure 130 for mechanically retaining a support matrix. In one embodiment, organoid cells can be mixed with a support matrix that provides a three-dimensional scaffold. In some embodiments, the organoid cells are mixed with the support matrix using the organoid culture dome method. Generally, the support matrix is similar to the extracellular matrix, such as a hydrogel that supports the growth and differentiation of organoids. Example support matrices that can be used are Substrate. In one exemplary method, a support substrate can be placed on ice to maintain the support substrate at a low temperature of approximately 0 °C, and then droplets of suspended cells in the range of about 5 microliters to about 50 microliters can be dispersed within the boundaries of the holding structure 130. The size of the holding structure 130 can be selected based on the desired size of the organoid dome, as discussed in more detail below. Additionally, other support substrate materials can be used. Droplets of similar size are set in the holding structures 130 adjacent to each other within a well 102 and 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 heated to approximately 37 °C before the droplets are dispersed on 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 and the height and diameter of the holding structure 130 within each well, as discussed in more detail below. Once the cell and support substrate mixture is dispersed on the bottom surface 118 of each well 102, the support substrate can be incubated at 37 °C to allow the gel to polymerize and form a dome structure. In some embodiments, the support substrate can gel in about 5 to about 10 minutes, and then the gelled material becomes a semi-solid dome structure in which the organoid cells are embedded. A culture medium containing niche factors can be applied to the polymerized dome such that organoids form and grow. As used herein, the term "analytical sample" refers to the droplet of cells in the support substrate that forms the organoid dome structure.

[0045] In an embodiment, each well 102 can include a single holding structure 130. In some embodiments, each well can contain multiple holding structures 130. In certain embodiments, each well 102 can include between approximately 1 and 50 holding structures. For example, the wells of a 24-well plate can contain 5 to 7 holding structures 130 at the bottom 116 of each well, while the 6-well plate can contain 20 to 30 holding structures 130 at the bottom 116 of each well. The holding structure 130 mechanically holds the droplet of cells suspended in a scaffold material that is similar to the extracellular matrix (also referred to as the support substrate), and the droplet of cells is placed within the boundaries defined by the holding structure 130, as described in more detail below.

[0046] As Figures 3 to 7 shown, each holding structure 130 can include a cavity 132 defined by a wall 134 and a cavity bottom surface 128, and provides a three-dimensional region to hold the droplet of the support substrate with suspended cells 180 that forms the dome structure. The size of the holding structure 130 of the microplate 100 and the number of wells 102 can be selected based on the desired size of the organoid dome. As Figures 3 to 6As shown, in some embodiments, the retention structure 130 can include a convex wall 136 and a cavity 132 that project from the bottom surface 118 of the hole 102. The convex wall 136 and the bottom surface 118 of the hole 102 can be integrally formed. In an embodiment, the convex wall 136 can be a continuous wall 138 having a closed perimeter or circumference. For example, the retention structure 130 can 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 bottom surface 128 of the cavity to the top surface 140 of the continuous wall 138. In an embodiment, the diameter (D1) can range from about 0.5 mm to about 5 mm. For example, in some embodiments, the diameter (D1) can range from about 0.5 mm to 2.5 mm, from about 2.5 mm to 5.0 mm, from about 0.5 mm to 1.0 mm, from about 1.0 mm to about 1.5 mm, from about 1.5 mm to about 2 mm, from about 2 mm to about 2.5 mm, from about 2.5 mm to about 3.0 mm, from about 3.0 mm to about 3.5 mm, from about 3.5 mm to about 4.0 mm, from about 4.0 mm to about 4.5 mm, or from about 4.5 mm to about 5.0 mm, including all ranges and sub-ranges therebetween.

[0047] In an embodiment, the height (H1) of the continuous wall 138 of the retention structure 130 can range from about 1 mm to about 3 mm. For example, in some embodiments, the height (H1) can range from about 1 mm to about 1.5 mm, from about 1.5 mm to about 2 mm, from about 2 mm to about 2.5 mm, or from about 2.5 mm to about 3 mm, including all ranges and sub-ranges therebetween. The height (H1) and the diameter (D1) can be selected based on the desired dome size to be formed by the dispensed cell droplets suspended in the support matrix 180. That is, the retention structure 130 can be configured to retain a specific droplet size in the range of about 5 microliters to about 50 microliters.

[0048] 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 still other embodiments, each holding structure can hold a droplet size of about 50 microliters. In certain embodiments, the droplet size can 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 sub-ranges therebetween.

[0049] The holding structure size can correspond to the number of wells 102 in each microplate 100, as described above. For example, typically 5 to 7 domes with an average volume of about 5 to about 10 microliters are produced and deposited on 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%, about 28% to about 30%, including all ranges and sub-ranges therebetween. In still other embodiments, the ratio of height (H1) to diameter (D1) can be about 30%. As stated above, the continuous wall 138 of the holding structure 130 can be configured to hold one of the analytical samples with a volume range between about 5 microliters and about 50 microliters deposited within the continuous wall 138. However, when the culture medium is added to the dome culture, the support matrix may swell in volume due to absorption of the culture medium. Thus, the volume of the organoid dome can increase to an expanded volume of 100 microliters. In some embodiments, the diameter of the organoid dome can be about 2 millimeters to about 4 millimeters. For example, the diameter of the organoid dome can 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 sub-ranges therebetween.

[0050] As Figure 6As shown, in an embodiment, the retention structure 130 may include a convex wall 136, where the convex wall 136 is a discontinuous wall or a set of discontinuous walls 142. Figure 6 Illustrates a three-dimensional region for retaining a droplet of a support matrix having suspended cells 180 that form a dome structure, but the droplet 180 is removed to show the discontinuous walls 142. The discontinuous walls 142 include an opening within the perimeter or circumference of the discontinuous walls. For example, the retention structure 130 may include a set of partial walls having a circular shape. Alternatively, the retention structure 130 may include a set of partial walls having an oval, polygonal, or other shape. Each retention structure 130 includes a diameter (D2) and a height (H2). In an embodiment, the diameter (D2) is the inner diameter measured from the inner wall of the retention structure 130. The height (H2) is measured from the bottom surface 128 of the cavity to the top surface 140 of the discontinuous wall 142. In an embodiment, the diameter (D2) may range between about 0.5 millimeters and about 5 millimeters. For example, in an embodiment, 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 millimeters and about 1.5 millimeters, 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.

[0051] In an embodiment, the height of the discontinuous wall 142 of the retention structure 130 can range from about 1 millimeter to about 3 millimeters. For example, in some embodiments, the 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 sub-ranges therebetween. The height (H2) and diameter (D2) can be selected based on the desired dome size formed by the dispensed cell droplets suspended in the support matrix. The ratio of the height (H2) to the diameter (D2) can range from about 10% to about 30%, such as in the range of 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 sub-ranges therebetween. The discontinuous wall 142 of the retention structure 130 can be configured to retain one of the analytical samples having a volume ranging from about 5 microliters to about 50 microliters deposited within the discontinuous wall 142. However, when the culture medium is added to the dome culture, the support matrix may swell in volume due to absorption of the culture medium. Thus, the volume of the organoid dome can increase to an expanded volume of 100 microliters or greater.

[0052] In an alternative embodiment, as Figure 7 illustrated, the retention structure 130 can include a cavity 132 without a convex wall. In such embodiments, the cavity 132 defined by the cavity wall 134 and the cavity bottom surface 128 provides a three-dimensional region to retain the droplet of the support matrix with suspended cells 180 that forms the dome structure. In such embodiments, the retention structure 130 includes a diameter (D3) and a height (H3). In an embodiment, the diameter (D3) is the inner diameter measured from the inner wall 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 hole 102. In an embodiment, the diameter (D3) can range from about 0.5 millimeters to about 5 millimeters.

[0053] For example, in some embodiments, the diameter (D3) can range from about 0.5 millimeters to about 2.5 millimeters, from about 2.5 millimeters to about 5.0 millimeters, from about 0.5 millimeters to about 1.0 millimeters, from about 1.0 millimeters to about 1.5 millimeters, from about 1.5 millimeters to about 2 millimeters, from about 2 millimeters to about 2.5 millimeters, from about 2.5 millimeters to about 3.0 millimeters, from about 3.0 millimeters to about 3.5 millimeters, from about 3.5 millimeters to about 4.0 millimeters, from about 4.0 millimeters to about 4.5 millimeters, or from about 4.5 millimeters to about 5.0 millimeters, including all ranges and sub - ranges therebetween.

[0054] In an embodiment, the height of the cavity wall 134 of the holding structure 130 can range from about 0.5 millimeters to about 3 millimeters. For example, in an embodiment, the height (H3) 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, from about 2.5 millimeters to about 3 millimeters, including all ranges and sub - ranges therebetween. The height (H3) and diameter (D3) can be selected based on the desired dome size formed by the dispensed cell droplets suspended in the support matrix. The ratio of the height (H3) to the diameter (D3) can range from about 5% to about 30%. The cavity 132 of the holding structure 130 can be configured to hold one of the droplets of a volume of the support matrix with suspended cells 180 that form a dome structure deposited within the cavity 132, wherein the volume can range from about 5 microliters to about 50 microliters. In certain embodiments, the volume range can be from about 5 microliters to about 10 microliters, from about 10 microliters to about 15 microliters, from about 15 microliters to about 20 microliters, from about 5 microliters to about 25 microliters, from about 20 microliters to about 25 microliters, from about 25 microliters to about 30 microliters, from about 30 microliters to about 35 microliters, from about 25 microliters to about 50 microliters, from about 35 microliters to about 40 microliters, from about 40 microliters to about 45 microliters, or from about 45 microliters to about 50 microliters, including all ranges and sub - ranges therebetween. However, it should be understood that the support matrix may expand in volume due to absorption of the culture medium. Thus, the volume of the organoid dome can increase to an expanded volume of 100 microliters or greater. For example, the organoid dome can increase to an expanded volume from about 10 microliters to about 200 microliters, from about 10 microliters to about 50 microliters, from about 50 microliters to about 100 microliters, from about 100 microliters to about 150 microliters, or from about 150 microliters to about 200 microliters (including all ranges and sub - ranges therebetween).

[0055] In other embodiments, such asFigure 8 As shown, the retention structure 130 can include a plurality of radially spaced-apart struts 150, each of the plurality of radially spaced-apart struts 150 forming a group. It should be understood that each hole 102 can include a plurality of retention structures 130. For example, in Figure 8 shown, seven (7) retention structures 130 are shown, each retention structure including a plurality of radially spaced-apart struts 150. Each group of struts 150 forming the retention structure 130 can alternatively form an oval, polygon, or other shape. In an embodiment, the struts 150 can each be cylindrical. However, other shapes are possible, including oval or polygon, or including but not limited to triangular, square, or rectangular. In an embodiment, the struts 150 of each retention structure 130 can surround a cavity, such as cavity 132, within the bottom 116 of the hole 102, as Figure 7 shown. In other embodiments, the struts 150 can be disposed on the bottom 116 of the hole 102 that does not have a cavity. The retention structure 130 of the struts 150 can include 3 to 4 struts. However, additional struts 150 can be used to form the retention structure 130. Each retention structure 130 of the struts 150 includes a diameter (D4). The diameter (D4) is the inner diameter measured from the inner wall 152 of the strut 150. In an embodiment, the diameter (D4) can range between about 0.5 millimeters and about 5 millimeters.

[0056] For example, in an embodiment, the diameter (D4) can 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 millimeters and about 1.5 millimeters, 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 sub-ranges therebetween.

[0057] Each strut includes a height (H4) that can range between about 1 millimeter and about 6 millimeters. For example, in some embodiments, the height (H4) can 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 hole 102. Thus, if the bottom 116 of each hole 102 includes a cavity, the height (H4) of the strut 150 may be shorter than the height of the bottom 116 of each hole 102 without the cavity. The strut 150 can be arranged to hold one of the droplets of the support matrix with suspended cells 180 having a volume ranging between about 5 microliters and about 50 microliters within the radially spaced struts 150. The ratio of the height (H4) to the diameter (D4) can range from about 10% to about 30%, such as 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 sub-ranges therebetween. Each strut 150 of the holding structure 130 can be configured to hold one of the analytical samples having a volume ranging between about 5 microliters and about 50 microliters deposited within the holding structure 130. However, when the culture medium is added to the dome culture, the support matrix may expand in volume due to absorption of the culture medium. Thus, the volume of the analytical sample can be increased to an expanded volume of 100 microliters or greater.

[0058] Figure 5 and 6 the convex wall 136 shown in Figure 7 the cavity wall 134 shown in Figure 8 and the strut 150 shown in have a relatively short height (H) compared to the diameter (D) of the holding structure 130 to avoid substantial interference with dome recovery while still (i) providing a physical barrier to protect the organoid dome from detachment due to liquid movement during medium exchange or drug treatment; and (ii) helping to maintain the shape and position of each organoid dome. The holding structure 130 can comprise an optically transparent material. In an embodiment, the holding structure 130 can be optically translucent. In some embodiments, the microplate can also be optically transparent or optically translucent.

[0059] In some embodiments, as Figure 9 shown, each retention structure 130 may include an inwardly chamfered edge 160 along the top portion 140 of the convex wall 136. The chamfered edge 160 may have an angle less than about 90 degrees, such as less than about 75 degrees, less than 65 degrees, less than 60 degrees, or less than 50 degrees. Alternatively, the retention structure 130 may include an inwardly beveled edge along the top portion 140 having an angle less than about 90 degrees, such as less than about 75 degrees, less than 65 degrees, less than 60 degrees, or less than 50 degrees. In other embodiments, the retention structure 130 may include a rounded edge. Fabricating the retention structure 130 with chamfered, beveled, or rounded edges may reduce the sharpness of the edges of the top portion 140 and improve the mechanical retention of the retention structure 130.

[0060] As Figure 10 shown, the microplate 100 may include a plurality of wells 102 that contain a retention structure 130a disposed on the bottom surface 118 of the well 102, the retention structure being in fluid communication with a set of retention structures 130b disposed on the bottom surface 118 of the well 102. In an embodiment, the retention structure 130a may be centrally disposed on the bottom surface 118 of the well 102, and the set of retention structures 130b is radially spaced from the retention structure 130a. In an embodiment, the retention structure 130a may be in fluid communication with the retention structure 130b via a set of fluid channels 170 that extend between the retention structure 130a and the set of second retention structures 130b to provide fluid communication therebetween. Each fluid channel 170 includes an inlet 172 for receiving cells suspended in a support matrix from the retention structure 130a and an outlet 174 for delivering the cells suspended in the support matrix to at least one of the retention structures 130b. Thus, multiple domes can be formed with a single pipette aspiration of a given volume of an assay sample containing cells suspended in a support matrix.

[0061] Turning Figure 11A to 11B , in an embodiment, the cavities 132 in the microplate 100 may be formed by a hot embossing process. In this process, the microplate 100 is fabricated by first softening the material of the bottom wall 112 of the microplate and then pressing the material against a mold 190. Thus, the shape of the mold 190 can be pressed and formed into the bottom wall 112 of the microplate. In some embodiments, the mold 190 may be in the shape of a polygonal column that is 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 oval cylinder. In some embodiments, the convex wall 136 may be formed from excess material that is pushed out of the cavity during the formation of the cavity 132. In some embodiments, the convex wall 136 may be a continuous wall 138 (asFigure 5 as shown in Figure 6 ; and in other embodiments, the convex wall 136 can be a discontinuous wall 142 (as shown in

[0062] . After hot embossing the cavities 132, the microplate 100 can be cooled. In some embodiments, the thickness of the bottom wall of the microplate 100 can range between about 0.05 millimeters and about 1 millimeter, between about 0.05 millimeters and about 0.5 millimeter, between about 0.5 millimeter and about 0.8 millimeter, or between about 0.8 millimeter and about 1.0 millimeter, including all ranges and sub - ranges therebetween. The bottom thickness of the hot - embossed cavity can range between about 0.02 and about 0.80 millimeters, between about 0.02 millimeters and about 0.1 millimeter, between about 0.1 millimeter and about 0.2 millimeter, or between about 0.2 millimeter and about 0.8 millimeter, including all ranges and sub - ranges therebetween.

[0062] In an embodiment, the microplate 100 having the cavities 132 can be formed by injection molding. In this process, the thermoplastic material of the microplate 100 is liquefied and then injected into the mold cavity of an injection molding machine. The mold cavity contains the shape of the microplate 100, which includes a plurality of holes 102 having cavities 132, as shown in Figure 7 . In some embodiments, the shape of the formed cavities 132 can be polygonal. In other embodiments, the shape of the formed cavities 132 can be cylindrical or elliptical. In some embodiments, the mold cavity can further contain the shape of the convex walls 136 that surround each of the cavities 132 (as shown in Figure 5 and Figure 11B ). In some embodiments, the convex wall 136 can be a continuous wall 138, and in other embodiments, the convex wall 136 can be a discontinuous wall 142 (as shown in Figure 6 ). Once the thermoplastic material is injected into the mold cavity, the injection molding machine cools the thermoplastic material to solidify the material and produce the microplate 100. Subsequently, the microplate can be removed. In some embodiments, the thickness of the bottom wall of the microplate 100 can range between about 0.05 millimeters and about 1 millimeter, between about 0.05 millimeters and about 0.5 millimeter, between about 0.5 millimeter and about 0.8 millimeter, or between about 0.8 millimeter to about 1.0 millimeter, including all ranges and sub - ranges therebetween. The cavity 132 can have a bottom wall thickness ranging between about 0.02 to about 0.80 millimeters, between about 0.02 millimeters and about 0.1 millimeter, between about 0.1 millimeter and about 0.2 millimeter, or between about 0.2 millimeter and about 0.8 millimeter (including all ranges and sub - ranges therebetween).

[0063] In an embodiment, the microplate 100 having a cavity 132 can be formed by casting. During this process, the thermoplastic material of the microplate 100 is liquefied and then introduced into the mold cavity. The mold cavity contains the shape of the microplate 100, and the microplate includes a plurality of holes 102 having a cavity 132, as Figure 7 shown. In some embodiments, the shape of the formed cavity 132 can be polygonal. In other embodiments, the shape of the formed cavity 132 can be cylindrical or elliptical. In an embodiment, the mold cavity can further contain the shape of a convex wall 136 that surrounds each of the cavities 132 (as Figure 5 and 11B shown). In some embodiments, the convex wall 136 can be a continuous wall 138, and in other embodiments, the convex wall 136 can be a discontinuous wall 142 (as Figure 6 shown). The liquefied thermoplastic material is solidified to produce the microplate 100. Subsequently, the microplate is removed. The thickness of the bottom wall of the microplate 100 can range from about 0.05 mm to about 1 mm, and the cavity 132 can include a bottom thickness ranging from about 0.02 to about 0.80 mm. In an embodiment, the thickness of the bottom wall of the microplate 100 can range from about 0.05 mm to about 0.5 mm, from about 0.5 mm to about 0.8 mm, from about 0.8 mm to about 1.0 mm, from about 0.02 mm to about 0.1 mm, from about 0.1 mm to about 0.2 mm, or from about 0.2 mm to about 0.8 mm, including all ranges and sub-ranges therebetween.

[0064] Figure 12 Present an overview of the teachings for using the above-described microplate 100. Figure 12 The flowchart of Figure 12 illustrates the operations of method 200. According to step 202, a microplate 100 is provided that includes a bottom wall 112 that includes a plurality of holes 102 arranged in a linear array of rows and columns. Each hole 102 includes a bottom surface 118 for receiving an analytical sample 180 of suspended cells in a support matrix. The bottom surface 118 includes a plurality of retention structures 130 to provide mechanical retention for one of the analytical samples 180 within each retention structure 130, where each retention structure 130 includes a diameter that can range from about 0.5 mm to about 5 mm and a height, and where the ratio of the height (H1) to the diameter (D1) can be in the range of about 10% to about 30%.

[0065] For example, the diameter can range from about 0.5 millimeters to about 5.0 millimeters, such as between about 0.5 millimeters and about 1.0 millimeters, between about 1.0 millimeters and about 1.5 millimeters, 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 sub-ranges therebetween.

[0066] In some embodiments, the ratio of the height (H1) to the diameter (D1) of the holding structure 130 can 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 sub-ranges therebetween.

[0067] According to optional step 204, the microplate 100 can be heated before depositing the analysis sample 180 into each of the holding structures 130. The microplate 100 can be heated at a temperature in the range of about 36 °C to about 40 °C in an incubator. For example, the microplate 100 can be heated overnight at a temperature of 37 °C in an incubator. According to step 206, the analysis sample 180 can be deposited into each of the holding structures 130. According to step 208, the analysis sample 180 can then be incubated in each of the holding structures 130 to polymerize the support matrix. Subsequently, according to step 210, a medium containing niche factors can be overlaid on the analysis sample to form organoids in the support matrix held in each of the holding structures 130. Figure 12 Provided at step 212, 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, experiments can be performed on the organoids. For example, organoid analysis can be performed on the organoids, including but not limited to drug sensitivity analysis and growth and viability analysis, as well as other in vivo analyses such as RNA and DNA isolation, immunohistochemistry, and gene manipulation. In one embodiment, the organoids can be propagated and expanded by removing the support matrix and dissociating the cells enzymatically or mechanically. The organoids can then be restored to culture conditions.

[0068] Accordingly, the present disclosure provides a microplate having a holding structure and a method of using the same. The present disclosure encompasses many changes and modifications that can be made. Thus, although embodiments in the form of the microplate and its method of use have been shown and described, those skilled in the art will readily appreciate that various additional changes and modifications can be made without departing from the scope of the present disclosure.

Claims

1. A microplate comprising a plurality of wells for receiving an assay sample of cells suspended in a support matrix, each well of the plurality of wells comprising a bottom surface, the bottom surface comprising at least one retention structure to provide mechanical retention for 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 the ratio of the height to the diameter being in the range of about 10% to about 30%.

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

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

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

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

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

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

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

12. The microplate according to 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. The microplate according to claim 12, wherein the matrix comprises a total of 6 holes, 12 holes, 24 holes or 48 holes.

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

15. The microplate according to 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 according to claim 1, wherein the at least one retaining structure is optically transmissive.

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

18. The method according to claim 17, further comprising: culturing the assay sample within each of the retaining structures to polymerize the support matrix; and overlaying the assay sample with a culture medium containing a niche factor to form organoids in the support matrix retained within each of the retaining structures.

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

20. The method according to claim 17, further comprising heating the microplate before depositing the assay sample within each of the retaining structures.

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

22. The microplate according to claim 21, wherein the plurality of optically transmissive retaining structures are formed by molding grooves in the bottom of each of the plurality of holes.

23. The microplate analysis according to claim 21, wherein the plurality of optically transmissive retaining structures are formed by hot pin embossing grooves into the bottom of each of the plurality of holes.

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