Systems and methods for passaging organoids

Abstract

The microplate includes a culture well, a feeding well, and at least one separation channel therebetween. The culture well, the feeding well, and the separation channel each contain a first solution, and the culture well contains a target organoid. A method of using such a microplate includes sealingly engaging a pipette with the feeding well. While maintaining a seal between the pipette and the feeding well, at least a portion of the first solution is aspirated into the pipette. This causes the aspirated portion of the first solution to flow from the culture well through the separation channel into the feeding well. While maintaining a seal between the pipette and the feeding well, a second solution is injected from the pipette. The injected second solution flows from the feeding well through the at least one separation channel into the culture well.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is filed as a PCT international patent application on March 14, 2023, and claims priority to and the benefit of U.S. Provisional Application No. 63 / 320,459, filed March 16, 2022, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Introduction Organoids are collections of organ-specific cell types that develop from stem cells or organ progenitors and self-organize through cell sorting and spatially constrained lineage commitment similar to that in vivo. In addition, organoids exhibit several properties. These include having multiple organ-specific cell types, being able to recapitulate some specific function of the organ, and having cells that group together and organize spatially. Organoids are formed by using stem cells or progenitor cells cultured in a 3D medium, such as extracellular matrix hydrogels, commercially available as Matrigel or Cultrex BME. Organoid bodies are made by embedding stem cells in the 3D medium. When pluripotent stem cells are used to generate organoids, the cells are usually, but not always, able to form embryoid bodies. These embryoid bodies are then pharmacologically treated with patterning factors to encourage the formation of the desired organoid identity. Organoids have also been generated using adult stem cells extracted from the target organ and cultured in 3D medium. These steps are typically performed in vitro in microplates or well plates that are typically incubated, agitated, washed, aspirated, etc. under specific criteria to adequately culture the organoids while removing dead cells that are expelled from the organoids as part of the culture process. Summary of the Invention [Means for solving the problem]

[0003] Abstract In one aspect, the technology relates to a method for passage of target organoids in a microplate comprising a culture well, a supply well, and at least one separation channel fluidically connecting the culture well and the supply well, wherein the culture well, the supply well, and at least one separation channel each contain a first solution, and the culture well contains a target organoid, and the method includes: sealingly engaging a pipette with the supply well; while maintaining a seal between the pipette and the supply well, aspirating at least a portion of the first solution into the pipette, wherein the aspirated portion of the first solution flows from the culture well through the separation channel into the supply well during aspirating; and while maintaining a seal between the pipette and the supply well, injecting a second solution from the pipette, wherein the injected second solution flows from the supply well through the at least one separation channel into the culture well during injection.In one example, the supply well comprises a supply well axis, the pipette comprises a pipette axis, and the supply well axis and the pipette axis are not aligned during the sealing engagement of the pipette with the supply well. In another example, the method further comprises placing the microplate in a tilted position, and sealingly engaging the pipette with the supply well is performed while the microplate is in the tilted position.In another example, the first solution and the second solution are different.In yet another example, the first solution contains a first liquid component and at least one substantially dead cell released from the target organoid.In yet another example, sealingly engaging the pipette with the supply well comprises contacting the first solution with the pipette.

[0004] In another example of the above embodiment, the method includes disposing live cells in the culture well prior to sealingly engaging the pipette with the feeding well. In another example, the method includes introducing a first solution into the culture well, the separation channel, and the feeding well prior to aspirating at least a portion of the first solution into the pipette.

[0005] In another aspect, the technology relates to a microplate, the microplate including a body at least partially defining a culture well including a culture well mouth and a culture well base, a feeding well including a feeding well mouth and a feeding well base, the feeding well mouth including a substantially round feeding well mouth cross-section, and at least one separation channel interconnecting the culture well and the feeding well, and a sheet fixed to the body, the sheet at least partially defining the culture well, the feeding well, and the at least one separation channel. In one example, the feeding well base includes a feeding well base cross-section that is different from the feeding well mouth cross-section. In another example, the feeding well base cross-section is substantially rectangular. In yet another example, the feeding well includes an intermediate cross-section between the feeding well mouth cross-section and the feeding well base cross-section that is different from both the feeding well mouth cross-section and the feeding well base cross-section. In yet another example, the at least one separation channel includes a plurality of separation channels.

[0006] In another example of the above embodiment, the culture well comprises a plurality of culture wells, and the plurality of separation channels are interconnected to at least two of the plurality of culture wells. In another example, at least one separation channel has a maximum height dimension of about 50 μm. In yet another example, the at least one separation channel has a plurality of sides, and at least one of the plurality of sides is the sheet. In yet another example, the culture well is a plurality of culture wells, and the feed well is a plurality of feed wells.

[0007] In another example of the above aspect, the sheet is secured to the body at walls disposed between adjacent ones of the culture wells and the feeding wells. hi another example, the body is a unitary piece.

[0008] In another aspect, the present technology relates to a microplate including a plurality of culture wells, a plurality of feeding wells separated from the plurality of culture wells by a plurality of walls, means for forming a sealing engagement between at least one of the plurality of feeding wells and a pipette inserted into at least one of the plurality of feeding wells, at least one separation channel interconnecting a first culture well of the plurality of culture wells and a first feeding well of the plurality of feeding wells, and a sheet secured to the plurality of walls, the sheet at least partially defining the plurality of culture wells, the plurality of feeding wells, and the at least one separation channel.

[0009] The following drawings, which form part of this application, are illustrative of the described technology and are not intended in any way to limit the scope of the present disclosure as claimed, the scope of which is based on the appended claims. [Brief description of the drawings]

[0010] [Figure 1A] FIG. 1A and FIG. 1B show top and bottom perspective views of an organoid microplate. [Figure 1B] FIG. 1A and FIG. 1B show top and bottom perspective views of an organoid microplate.

[0011] [Diagram 2] 2A and 2B show partial cross-sectional views and enlarged partial cross-sectional views of the organoid microplate of FIGS. 1A and 1B.

[0012] [Diagram 3] 3A and 3B show enlarged bottom perspective and bottom views of the organoid microplate of FIGS. 1A and 1B.

[0013] [Figure 4] Figures 4A and 4B show cross-sectional views of an organoid microplate with a pipette inserted into a feeding well.

[0014] [Figure 5A-B] 5A-5C show alternative sealing elements for use with organoid plates. [Figure 5C] 5A-5C show alternative sealing elements for use with organoid plates.

[0015] [Figure 6] FIG. 6 shows a method for passaging organoids in organoid plates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description 1A and 1B show top and bottom perspective views of an organoid microplate 100, which will be described together. The microplate 100 (also referred to herein as a well plate) includes a top portion or body 102 and a base 104 made of a sheet material. As shown in FIG. 1, the top portion includes a body 102, which has multiple well units for growing, culturing, monitoring, and assaying embryoid bodies, fused embryoid bodies, spheroids, organoids, and / or other multicellular bodies. As used herein, the term "organoid" is used generally to refer to cells or cell aggregates grown from stem cells or pluripotent stem cells in a microplate, regardless of the specific growth stage or the specific type of cells or the number of cells grown. In various examples, the well plate body 102 is made of a material having a top surface 106 and a bottom surface 108. The microplate 100 may be characterized by a width W, a length L, and a height H. The components of the microplate body 102 may be formed from any suitable material and by any suitable process. In an example, the microplate body 102 may be formed solely or primarily from a polymer, such as a transparent polymer, and / or other recognizable materials. For example, the polymer may include one or more of polystyrene, polypropylene, poly(methyl methacrylate), cyclic olefin polymer, cyclic olefin copolymer, and / or other recognizable polymers. In an example, acrylonitrile butadiene styrene (ABS) may be utilized. The microplate body 102 may not have removable / movable parts and / or may be formed as a single integral piece, such as by injection molding or 3D resin printing, such that all of the structures (e.g., wells) of the microplate body 102 are integrally formed with each other. The base sheet material 104 may be secured to the bottom of the microplate body 102 as described herein, or may be integrally formed from the same material as the body 102. The materials used for the various components of the microplate 100 may be those that are compatible with each other and meet the required or desired performance characteristics of the microplate 100 .The resins used to form the body 102 of the microplate 100 and to adhere the sheet material 104 to its bottom exhibit acceptable adhesion when cured, resistance to flow when uncured, and resistance to liquid seepage from the wells. In addition to the materials listed above, other materials for use in the body 102 or sheet material 104 will be apparent to those skilled in the art. For example, an optically clear foil may be used for the sheet material 104.

[0017] Each well unit comprises a primary well compartment, also referred to as a primary or culture well 110, and a secondary well compartment, also referred to as a feed well or replenishment well 112. In various examples, the culture well 110 and the feed well 112 can be fluidly connected to each other to facilitate the flow of liquid (e.g., feed medium) between the culture well 110 and the feed well 112, as described herein. For example, the culture well 110 and the feed well 112 can be fluidly connected to each other via at least one separation channel (shown in Figures 3A and 3B below) sized and shaped to facilitate the flow of liquid medium between the two well compartments 110, 112. The exchange of medium between the culture well 110 and the feed well 112 removes toxic by-products and replenishes new nutrients to the growing cell culture. As used herein, the term "well unit" refers to a fluidly connected combination of wells including at least one culture well 110 and at least one feed well 112 linked by at least one separation channel. In examples, multiple culture wells 110 may be fluidly coupled to and fed by a single feeding well 112. In other examples, multiple feeding wells 112 may be coupled to a single culture well 110. In still other examples, multiple culture wells 110 and multiple feeding wells 112 may be coupled for liquid flow. As described in more detail below, separation channels are formed in one or more walls 114 that generally separate adjacent wells.

[0018] In an example, the culture wells 110 are sized and shaped to support deposited cell aggregates, which may be embedded in a hydrogel deposited at the base of each culture well 110. For example, each culture well 110 is used to grow embryoid bodies, fused embryoid bodies, spheroids, organoids, and / or other multicellular bodies, as may be recognized. According to various embodiments and depending on the number of well units in the microplate 100, the width of each culture well 110 may be up to about 8 millimeters (mm) (e.g., for a 96-well plate), up to 11 mm (e.g., for a 48-well plate), up to about 17 mm (e.g., for a 24-well plate), and / or other sizes, as may be recognized. In addition, the depths of the culture wells 110 and the feeding wells 112 are specified so that the microplate 100 can be tilted without spilling liquid from the respective culture wells 110 or feeding wells 112 of each well unit, as described below. 1B, the depth of each of the culture wells 110 and the feeding wells 112 is less than the total height H of the microplate 100, which may aid in stacking multiple microplates 100 for storage. As can be seen in FIG. 1B, the base sheet material 104 is positioned so that it is slightly higher than the bottom surface 108 of the microplate 100.

[0019] The feeding well 112 may be used to replenish feeding medium and / or other nutrients that may be used to feed the growing cell aggregates positioned in the culture well 110. In addition, the feeding well 112 may be used to harvest supernatant from the cell aggregates, as may be appreciated. For example, the feeding well 112 may be used to introduce feeding medium and / or other nutrients that may be used by the growing cell culture in the culture well 110. The feeding well 112, according to various embodiments of the present disclosure, is sized and shaped to hold liquid that may be exchanged to and from the culture well 110. According to various embodiments and depending on the number of well units in the microplate, the width of the feeding well 112 may be up to about 8 millimeters (mm) (e.g., for a 96-well plate), up to 11 mm (e.g., for a 48-well plate), up to about 17 mm (e.g., for a 24-well plate), and / or other sizes that may be appreciated.

[0020] The size and shape of the culture well 110 and the feeding well 112 may differ from each other. In some examples, the culture well 110 is larger (in a dimension, e.g., diameter or volume) than the feeding well 112. In other examples, the feeding well 112 is larger than the culture well 110. In some examples, the culture well 110 has a shape different from that of the feeding well 112. The well units are preferably arranged in columns and rows as shown in FIG. 1A. In various embodiments, the microplate 100 comprises a 90-size (96) well-type plate with 96 main well compartments for cell culture, as can be appreciated. However, it should be noted that the microplate 100 is not limited to a 96-well type plate, and can be organized as a strip or other type of configuration, as can be appreciated. Additional details regarding the configuration of the feeding well 112 are provided below.

[0021] 2A and 2B show partial cross-sectional views and enlarged partial cross-sectional views of the organoid microplate 100 of FIG. 1A and FIG. 1B. Certain elements shown in FIG. 2A and FIG. 2B have been described above in the context of FIG. 1A and FIG. 1B and will not be described further. FIG. 2A and FIG. 2B are described simultaneously and not all features or elements of one view are shown in the other. The cross-sectional views of FIG. 2A and FIG. 2B generally reveal the interior of the feed channel 112. In addition, the lower end 116 of the wall 114 is shown and its height relative to the bottom surface 108 of the microplate 100 is evident. The feed well 112 is formed with a unique configuration that allows for certain performance. Some advantages of the configuration of the feed well 112 include ensuring a reliable seal between the feed well 112 and a pipette inserted therein, ensuring proper flow between the feed well 112 and the associated culture well 110, and ensuring unimpeded liquid flow within the feed well 112. The supply well 112 may be defined by a supply well axis Aw.

[0022] Each of the feed wells 112 includes a feed well mouth 118 disposed near the top surface 106 of the microplate 100. In an example, the feed well mouth 118 has a substantially round cross-section, and in an example, the feed well mouth 118 may be circular, oval, elliptical, or defined by a primarily curved perimeter. Furthermore, the feed well mouth 118 may be centered on a well axis Aw. The substantially round cross-sectional shape of the feed well mouth 118 generally corresponds to the outer shape of a pipette (not shown), which allows for sealing engagement of the feed well 112 with a pipette when inserted, as described further below. In the illustrated example, the feed well 112 also includes a feed well base 120, which has a cross-sectional shape different from that of the feed well mouth 118. The shape of the feed well base 120 is substantially rectangular, and is also substantially centered on the well axis Aw. In other examples, the shape may be defined as a substantially rectangular shape with rounded corners, a square, a rounded squircle, or some other shape. Shapes that match the shape of the feed well orifice are also contemplated. However, as described in more detail below, a feed well base 120 having at least one straight or substantially straight side may be desirable to ensure desired liquid flow through the separation channel. At an intermediate location 122 between the feed well orifice 118 and the feed well base 120, the cross-sectional shape of the feed well 112 may differ from the cross-sectional shapes of both the feed well orifice 118 and the feed well base 120 as the cross-sectional shape of the feed well 112 transitions from one to the other. It may be desirable for the walls of the feed well 112 to smoothly transition from one shape to another to avoid eddies or other features that may impede the flow of liquid, debris, etc. from the culture well 110. FIG. 2B shows a culture well orifice 124 having a substantially hexagonal cross-sectional shape, although other shapes are contemplated.

[0023] 3A and 3B show a partially enlarged perspective bottom view and a partially enlarged bottom view of the organoid microplate 100 of FIG. 1A and FIG. 1B, respectively. Certain elements shown in FIG. 3A and FIG. 3B have been described above in the context of other figures and will not be described further. FIG. 3A and FIG. 3B are described simultaneously and not all features or elements of one figure are shown in the other. A plurality of separation channels 126 are formed at the lower end 116 of the wall 114 that separates the culture well 110 of the well unit from the associated feed well 112. In the illustrated figure, four separation channels 126 are shown, although more or less such channels 126 may be utilized. In cross section along the length of the separation channels 126, each may have a cross-sectional profile of any desired or required shape, although a substantially rectangular shape may be desirable to maintain the desired flow therethrough. In the illustrated example, the material of the microplate body 102 forms the top surface and two side surfaces of each separation channel 126. The sheet material (not shown) when secured to the lower end 116 of the wall 114 of the body 102 defines the bottom surface of each separation channel 126. The dimensions of the separation channels 126 may be as required or desired for a particular application. In an example, the height of the separation channels 126 may be about 10 μm, about 25 μm, or about 50 μm. In an example, a height of up to about 200 μm may be utilized to feed large organoids in the culture well 110. For single cell applications, separation channels 126 having heights of less than about 10 μm, such as less than about 5 μm or less than about 3 μm, may be utilized. The width of the separation channels 126 may also vary from application to application. For example, a single wide separation channel may be utilized between the feed well 112 and the culture well 110. Several separation channels 126 using rectangular, square, or round shapes may be utilized, with the width being equal to or greater than the height of the channel. The number of channels may also vary. Depending on the size of the wells and the particular application, one, two, three, four, or more channels may be used. The channels may be formed from the same molded plastic of the microplate 100.Alternatively, a very thin foil, similar to the foil used to form the base of a microplate, may be used to form the separation channel 126. In an example, a 135 μm thick foil may be utilized.

[0024] FIG. 3B more clearly depicts the cross-sectional shape of the base of the culture well 110 as substantially hexagonal. Although other shapes are contemplated, it may be advantageous to have a uniform or substantially uniform shape along the entire height of the culture well 110 to allow easy access to, removal of, and the like from, the cell aggregates within the well. A substantially rectangular cross-sectional shape of the feed well base 120 is also depicted. A separation channel 126 fluidly connects the two wells 110, 112, in this example between the substantially straight adjacent sides of the wells 110, 112. While each separation channel 126 need not be identical in length (or shape), maintaining substantially similar dimensions may help ensure desirable liquid transfer between the wells 110, 112 without leaving residual liquid in one channel 126 while another channel 126 is drained to an empty state.

[0025] The process of removing debris (e.g., dead or partially dead cells) from organoid cultures is commonly referred to as passaging. In the microplate 100 shown herein, as well as in similarly configured microplates according to the teachings herein, passaging is performed by creating a liquid flow between each culture well 110 and its associated feeding well 112. This may be required at several specific times during the organoid formation process. By introducing and removing liquid from the feeding well 112, rather than directly from the culture well 110, several advantages are obtained. One advantage is that the separation channel 126 in between acts as a filter to prevent live (and generally larger) cells and aggregates from being drawn off along with the liquid and the dead cells being removed. Another advantage is that there is no accidental contact between the pipette and the live cells and organoids (and hydrogels) in that the pipette is not inserted into the culture well 110 until its removal is specifically required. Other advantages are described below.

[0026] In known microplates that contain only culture wells (without the feeding wells described herein), passaging typically involves placing such microplates in a centrifuge for a spinning operation. This operation forces the organoids, large fractions, and live cells to a location in the culture well 110 away from dead cells, which may form a top layer or otherwise be suspended in the liquid in the culture well. This top layer of liquid, including debris (e.g., dead cells), must then be removed, followed by the introduction of new clean liquid, if applicable in the process. Inserting a pipette into the cultured cells for such removal requires fine positioning of the pipette to prevent accidental removal of organoids or other desired cells or aggregates.

[0027] However, the microplates described herein and those manufactured according to the teachings herein include a feed well that can be used to reduce accidental removal of organoids and otherwise improve the organoid formation process. Instead of placing the microplates described herein in a centrifuge, in an example, the present technology also utilizes gravity for separation. Returning to the diagrams described above, the microplate 100 can be tilted or tilted, thereby collecting the organoids and heavy live cells and fractions away from the separation channel 126. A pipette is then inserted into the feed well 112 and the liquid therein is aspirated. This aspirate draws the liquid from the culture well 110, through the separation channel 126, into the feed well 112, and into the pipette. The lighter dead cells and other debris suspended in the liquid are removed with it, while the heavier organoids, live cells, and aggregates remain because the separation channel 126 acts as a filter for such large products. Then, new liquid or other solution may be introduced into the supply well 112, and the new liquid or other solution may flow by gravity to equilibrium with the culture well 110. However, this flow by gravity may be undesirably slow for some applications. In an example, due to the size and number of the separation channel, the liquid introduced by gravity into the supply well 112 may take up to one minute to flow into the culture well 110 and reach equilibrium. The specific structural features of the microplate 100 described herein, as well as similar configurations that will be apparent to those skilled in the art, further improve the aspiration and injection steps that are performed as part of the organoid passaging. These steps are described below, first in the context of Figures 4A and 4B.

[0028] 4A and 4B show a cross-sectional view of the organoid microplate 100 with a pipette 200 inserted into the feeding well 112. The pipette 200 may be inserted into the feeding well 112 and penetrate to a depth where the pipette 200 contacts liquid that may be present in the feeding well 112. However, this contact is not required for aspiration or injection of liquid. This is because the feeding tube mouth 118 is substantially round and therefore forms a sealing engagement with the pipette 200 itself, which is also substantially round. Due to this sealing engagement, during aspiration, liquid is drawn from the culture well 110 through the separation channel 126, into the feeding well 112, and into the pipette, which is under negative pressure. When injecting liquid, the sealing engagement allows the positive pressure introduction of liquid from the pipette and thus the passage of liquid into the feeding well 112 and through the separation channel 126 into the culture well 110. In examples, flow rates of up to about 80 μL / sec, up to about 90 μL / sec, up to about 95 μL / sec, up to about 100 μL / sec, up to about 105 μL / sec, and up to about 110 μL / sec may be achieved, thereby significantly reducing operation times. In FIG. 4A, the well axis Aw and the pipette Ap may be substantially aligned as shown.

[0029] The configuration of the microplate 100 with a substantially round feed well mouth 118 that is in sealing engagement with the pipette allows for further advantages, one of which is illustrated in FIG. 4B. Here, the microplate 100 is tilted as part of the organoid passaging process described above. The sealing engagement between the substantially round feed well mouth 118 and the substantially round pipette 200 may be maintained while tilting the microplate at tilt angles of, for example, up to about 2°, up to about 3°, up to about 4°, up to about 5°, up to about 6°, up to about 7°, or more. Under these conditions, the well axis Aw and the pipette Ap do not align as shown in FIG. 4B. This may be particularly advantageous, as aspirating while maintaining the tilt of the microplate 100 may help keep organoids and live cells away from the separation channel and reduce accidental aspiration thereof. Furthermore, it should be noted that automated pipette systems, which include multiple pipettes, typically in a row, are typically mechanically actuated (raised and lowered) only in the vertical direction. Thus, a microplate 100 as described herein utilizing structures capable of sealing engagement with a pipette may still be utilized with such an automated system while the microplate 100 remains in a tilted configuration.

[0030] 5A-5C show alternative sealing elements for use with the organoid microplate 100. Generally, each of these sealing elements is used to place a pipette into sealing engagement with the feeding wells 112 of the microplate 100. In the illustrated example, the sealing element is positioned adjacent to the feeding well mouth 118 during the aspiration or injection step. FIG. 5A shows a sealing element including a gasket 500, which may be secured near the periphery of the feeding well mouth 118, for example, by adhesive or other fasteners. In other examples, the gasket may instead be secured to the pipette and contact the feeding well mouth 118 to form a sealing engagement. FIG. 5B shows a resilient or flexible septum 502, which may span the entire feeding well mouth 118. The septum 502 may define an opening 504 of sufficient diameter to receive and close around the end of the pipette 200 when inserted. A single sheet may be formed with a configuration of septa sufficient to cover the top surface of the microplate 100, with appropriate openings 504 formed in the sheet as well as openings allowing access to the culture wells. The sheet may then be adhered to the top surface of the microplate 100.

[0031] 5C shows an elongated throat 508 that may extend from the feed well opening 118. The throat 508 may extend above the top surface and may be tapered or otherwise shaped to match the taper of the pipette 200. The taper need not be an exact match, merely sufficient to provide adequate contact with the exterior surface of the pipette 200 to form a sealing engagement upon insertion. Another example of a sealing element contemplates that the top portion of the microplate is formed of a more flexible or resilient material than the remainder of the microplate, allowing that portion to conform more easily to the pipette.

[0032] FIG. 6 illustrates a method 600 for passaging a subject organoid in a microplate. The microplate may be configured as described herein or may otherwise follow the teachings herein, as will be apparent to one of skill in the art upon reading this disclosure in its entirety. In that regard, the microplate may include at least one culture well, at least one feeding well, and at least one separation channel fluidically connecting the culture well and the feeding well. In many applications, the microplate includes multiple culture wells fluidically connected to multiple feeding wells via multiple separation channels. Additionally, a substrate such as a hydrogel may be placed in the culture well to support the live cells, organoids, etc. during the formation process. The method 600 may begin with two optional operations. In operation 602, a first liquid solution is introduced into the culture well, the separation channel, and the feeding well. The liquid solution may be introduced into the feeding well or the culture well until a desired liquid equilibrium is achieved. In optional operation 604, live cells may be placed on the substrate in the culture well by known methods. Operations 602 and 604 may be performed in the order shown, in reverse order, or substantially simultaneously. At any point during the organoid formation process, the microplate may be tilted (operation 606) to collect the desired contents (e.g., live cells, organoids, etc.) away from the separation channel.

[0033] Method 600 continues with operation 608, sealingly engaging the pipette with the feeding well. As described elsewhere herein, sealing engagement may occur when the pipette contacts the feeding well mouth to form a seal. Other examples are described herein, such as a seal formed by contact with a gasket, a septum, or an elongated throat shaped to sealingly engage with the pipette. If optional operation 606 is performed, a feeding well axis defining the feeding well will not be aligned with a pipette axis defining the pipette during the sealing engagement. Depending on the height of liquid disposed within the feeding well, the length and other dimensions of the feeding well, and the insertion depth of the pipette into the feeding well, the first solution may be contacted when sealing engagement is made (operation 610). Additionally, operations 608 and 610 may be performed substantially simultaneously with operation 606, in which case the microplate is maintained in a tilted position during sealing engagement of the pipette with the feeding well, thereby preventing accidental aspiration of organoids, live cells, etc. during subsequent operations.

[0034] While the sealing engagement is maintained, an operation 612 is performed, in which at least a portion of the first solution is aspirated into the pipette. During this aspiration, the first solution flows from the culture well through the separation channel and into the feeding well during aspiration, thereby being drawn into the pipette. In an example, the aspirated first solution may contain a first liquid component and at least one substantially dead cell released from the target organoid. At this point, the pipette may be removed from the sealing engagement with the feeding well, thereby allowing for disposal of the aspirated first solution. Thereafter, in an operation 614, a second solution may be injected from the pipette while maintaining a seal between the pipette and the feeding well. The first and second solutions may be different. In another example, the second solution may be the same as the first solution, thereby causing turbulent agitation of the solution to help release debris, such as dead cells, from the organoid. Once sufficient agitation has occurred, the agitated solution and its contents may be aspirated and disposed of. The injected second solution flows from the supply well through at least one separation channel into the culture well during injection. The pipette used for injection operation can be the same as the pipette used for aspirating operation, or a different pipette can be used according to the best practice of the laboratory where the method 600 is performed, for example, to prevent cross-contamination. Thus, in the context of the method 600 described herein, the term "pipette" should not be considered to be limited to a single same pipette. In an example, the aspirated first solution can contain the first liquid component and at least one substantially dead cell released from the target organoid.

[0035] It should be understood that the present disclosure is not limited to the specific structures, process steps, or materials disclosed herein, but extends to equivalents thereof that would be recognized by one of ordinary skill in the art. It should also be understood that the terminology used herein is used merely for the purpose of describing specific examples, and is not intended to be limiting. It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly requires otherwise.

[0036] It will be apparent that the systems and methods described herein are well adapted to achieve the stated objects and advantages as well as those inherent therein. Those skilled in the art will recognize that the methods and systems herein may be implemented in numerous ways and are therefore not limited by the illustrated examples and examples set forth above. In this regard, any number of the features of the various examples described herein may be combined into one example, and alternative examples having fewer or more than all of the features described herein are possible.

[0037] While various examples have been described for purposes of this disclosure, various changes and modifications may be made that are well within the scope contemplated by this disclosure. Numerous other modifications may be made that will readily occur to those skilled in the art and are encompassed by the spirit of this disclosure.

[0038] The following is claimed:

Claims

1. 1. A method of passaging a subject organoid in a microplate comprising a culture well, a feeding well, and at least one separation channel fluidly connecting said culture well and said feeding well, wherein said culture well, said feeding well, and said at least one separation channel each contain a first solution, and said culture well contains said subject organoid, said method comprising: sealingly engaging a pipette with said supply well; aspirating at least a portion of the first solution into the pipette while maintaining the seal between the pipette and the feeding well, wherein an aspirated portion of the first solution flows from the culture well, through the separation channel and into the feeding well during aspiration; injecting a second solution from the pipette while maintaining the seal between the pipette and the supply well, wherein the injected second solution flows from the supply well through the at least one separation channel and into the culture well during injection.

2. 2. The method of claim 1, wherein the supply well comprises a supply well axis and the pipette comprises a pipette axis, the supply well axis and the pipette axis being misaligned during the sealing engagement of the pipette with the supply well.

3. 3. The method of claim 2, further comprising placing the microplate in a tilted position, and wherein sealingly engaging the pipettes with the supply wells occurs while the microplate is in the tilted position.

4. The method of any one of claims 1 to 3, wherein the first solution and the second solution are different.

5. 5. The method of claim 4, wherein the first solution contains a first liquid component and at least one substantially dead cell released from the target organoid.

6. The method of any of claims 1 to 5, wherein sealingly engaging the pipette with the supply well comprises contacting the first solution with the pipette.

7. The method of any of claims 1 to 6, further comprising disposing live cells in the culture well prior to sealingly engaging the pipette with the supply well.

8. 8. The method of claim 7, further comprising introducing the first solution into the culture well, the separation channel, and the supply well prior to aspirating at least a portion of the first solution into the pipette.

9. A microplate comprising: A main body, a culture well having a culture well mouth and a culture well base; a supply well comprising a supply well mouth and a supply well base, said supply well mouth comprising a substantially round supply well mouth cross-section; and At least one separation channel interconnecting said culture well and said supply well. a body at least partially defining a sheet secured to the body, the sheet at least partially defining the culture well, the feeding well, and the at least one separation channel; A microplate comprising:

10. 10. The microplate of claim 9, wherein the feeding well base comprises a feeding well base cross-section that is different from the feeding well mouth cross-section.

11. The microplate of claim 10, wherein the feeding well base cross-section is substantially rectangular.

12. 12. The microplate of claim 10, wherein the feeding well comprises an intermediate cross-section between the feeding well mouth cross-section and the feeding well base cross-section, the intermediate cross-section being different from both the feeding well mouth cross-section and the feeding well base cross-section.

13. The microplate of any of claims 9 to 12, wherein the at least one separation channel comprises a plurality of separation channels.

14. 14. The microplate of claim 13, wherein the culture well comprises a plurality of culture wells, and the plurality of separation channels are interconnected to at least two of the plurality of culture wells.

15. The microplate of any one of claims 9 to 14, wherein the at least one separation channel has a maximum height dimension of about 50 μm.

16. The microplate of any one of claims 9 to 15, wherein the at least one separation channel comprises a plurality of sides, and at least one of the plurality of sides comprises the sheet.

17. The microplate of any one of claims 9 to 16, wherein the culture well comprises a plurality of culture wells and the supply well comprises a plurality of supply wells.

18. 18. The microplate of claim 17, wherein the sheet is secured to the body at walls disposed between adjacent ones of the culture wells and the feeding wells.

19. The microplate of any one of claims 9 to 18, wherein the body comprises a single piece.

20. A microplate comprising: A plurality of culture wells; a plurality of feeding wells separated from said plurality of culture wells by a plurality of walls; means for forming a sealing engagement between at least one of the plurality of supply wells and a pipette inserted into at least one of the plurality of supply wells; at least one separation channel interconnecting a first culture well of the plurality of culture wells and a first supply well of the plurality of supply wells; a sheet secured to the plurality of walls, the sheet at least partially defining the plurality of culture wells, the plurality of feeding wells, and the at least one separation channel.

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