Systems and methods for organoid culture

Abstract

The present disclosure provides a system for culturing, monitoring, and / or analyzing organoids, including methods and apparatus. In an exemplary method of organoid culturing, the method can include disposing a scaffold in a receptacle having an open side. A sealing member can be coupled to the open side of the receptacle to form a chamber. The scaffold can be used to form an organoid in the chamber. Fluid and / or at least one substance can be introduced from an overlying reservoir into the chamber to contact the organoid.

Description

[0001] This application is a divisional application of patent application No. 2020800422416, filed on May 7, 2020, entitled "System and Method for Organoid Culture".

[0002] Related applications

[0003] This application claims priority to U.S. Provisional Application No. 16 / 407,026, filed May 8, 2019, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0004] Organoids (“small organoids”) are three-dimensional clumps of different types of cells generated in vitro and share some similarities with organs, such as exhibiting the actual histology of organ-specific tissues. Clumps of cells can be generated by seeding a matrix with a small number of stem cells. The stem cells then proliferate, differentiate, and self-organize within the matrix, using the matrix as a scaffold. Through this method, organoids resembling tissues from the brain, heart, intestine, kidney, liver, and stomach have been generated to date. These promising results suggest that organoid culture has the potential to provide new insights into organ development and function, and may be able to reproduce disease models that allow for in vitro drug screening. Organoids could revolutionize how drugs are discovered and how drugs are personalized.

[0005] Several challenges limit researchers' ability to fully develop organoids. First, as organoid size increases, they may require feeding from both the inside and outside, presenting a challenge. Second, each different organoid type requires an optimized three-dimensional matrix scaffold structure, a culture medium exchange (feeding) suitable for that structure, and may even need the ability to withstand mechanical resistance or controlled forces to allow for proper growth and development of functional organoids. Third, there are no containers optimized for exposing large organoids to different types of reagents to allow for screening. Fourth, there are no containers optimized for in-situ monitoring of large organoids using imaging methods. Instead, organoids are typically imaged after they have been fixed and physically sliced. Therefore, there is a need for systems and methods to improve organoid culture, such as by providing the ability to efficiently grow, monitor, and analyze large organoids. Summary of the Invention

[0006] This disclosure provides systems, methods, and apparatus for culturing, monitoring, and / or analyzing organoids or other organized multicellular structures. In an exemplary method of organoid culture, the method may include disposing a scaffold in a receiver having an open side. A sealing member may be incorporated into the open side of the receiver to form a chamber. The scaffold may be used to form the organoid within the chamber. Fluid and / or at least one substance may be introduced from an overlying reservoir into the chamber to contact the organoid. Attached Figure Description

[0007] Figure 1 This is a schematic side view of an exemplary container for forming, culturing, monitoring, and analyzing organoids, wherein the container contains organoids immersed in a liquid culture medium, and the front and rear walls of the container are not visible.

[0008] Figure 2 yes Figure 1 The exploded side view of the container when the container is empty.

[0009] Figure 3 yes Figure 2 A side view of the main body of the container, which is as follows Figures 4-10 The methods for culturing, monitoring, and / or analyzing organoids in the containers shown are plotted separately and relative to the actual process before execution. Figure 2 Inverted.

[0010] Figure 4 and Figure 5 yes Figure 3 The side view of the container body, drawn during and after the formation of the scaffold in the container's receiver by 3D printing or pipetting.

[0011] Figure 6 yes Figure 5 A side view of the container body, drawn after the prefabricated sealing member has been attached to the container body to seal the opening side of the receiver, thereby creating a chamber containing the support.

[0012] Figure 7 yes Figure 6 A side view of the container body and sealing components, drawn after the container body has been flipped into an organoid culture orientation, at least one liquid culture medium has been introduced into a chamber containing a scaffold and into an overlying reservoir in fluid communication with the chamber, and the container lid has covered the top of the reservoir opening.

[0013] Figure 8 It was drawn after stem cells proliferated and differentiated within the scaffold to produce organoids. Figure 7 A side view of the container.

[0014] Figure 9 It was drawn after the scaffold was reshaped / replaced by inducing organoid development. Figure 8 A side view of the container.

[0015] Figure 10 These images were created when organoids were imaged using light-sheet microscopy. Figure 9 A side view of the container.

[0016] Figures 11-13yes Figure 5 A side view of the container body and support, which shows an alternative method of forming a chamber by sealing the opening side of the receiver of the container body by forming a sealing member in situ in the receiver.

[0017] Figure 14 yes Figure 13 A side view of the container body, sealing member, and support, drawn with an imaging objective and after the container body has been flipped into an organoid culture orientation, at least one liquid culture medium has been introduced into a chamber containing the support and into an overlying reservoir in fluid communication with the chamber, and the container lid has covered the top of the opening of the reservoir.

[0018] Figure 15 yes Figure 12 A side view of the container body and support, showing relative to... Figure 13 An improved method is proposed, which seals the opening side of the receiver of the container body to form a chamber by forming a sealing member in situ in the receiver.

[0019] Figure 16 This is a schematic side view of an exemplary container strip for forming, culturing, monitoring and / or analyzing organoid arrays, wherein the container strip has a set of containers directly connected to each other.

[0020] Figure 17 yes Figure 16 A schematic side view of a container strip, drawn during organoid culture, and illustrating an exemplary pump configuration for transferring liquid culture medium between reservoirs within each container of the container strip.

[0021] Figure 17A and Figure 17B It shows Figure 16 A schematic side view of the container strips, drawn during organoid culture, and illustrating how tilted container strips in opposite directions of rotation can drive flow in their respective opposite directions within each chamber.

[0022] Figure 18 This is a schematic side view of another embodiment of an exemplary container strip with a container array, drawn during organoid culture and illustrating an exemplary pump configuration for transferring liquid culture medium between reservoirs of different containers in the container strip.

[0023] Figure 19 It is used for Figure 2 A view of an exemplary embodiment of the container body of the container.

[0024] Figure 20 yes Figure 19 A front view of the container body.

[0025] Figure 21 yes Figure 19 A view of the container body, drawn with the container body inverted and prefabricated sealing components separated from the receiver of the container body.

[0026] Figure 22 yes Figure 19 The main body of the container is roughly along Figure 20 The bottom view is taken from line 22-22.

[0027] Figure 23 yes Figure 19 The main body of the container is roughly along Figure 20 The top view of the cut-off line 23-23.

[0028] Figure 24 yes Figure 19 The main body of the container is roughly along Figure 23 The cross-sectional view taken from line 24-24.

[0029] Figure 25 yes Figure 19 The main body of the container is roughly along Figure 23 Another cross-sectional view taken from line 25-25.

[0030] Figure 26 Another embodiment of the container body is roughly as follows: Figure 25 Such a partial cross-sectional view, besides showing only the main body of the container, differs from... Figure 25 The lower part.

[0031] Figure 27 yes Figure 26 A partial cross-sectional view of the container body after porous tubes are formed between the channels of the container body through 3D printing.

[0032] Figure 28 It is another implementation of the container body, for example Figure 25 Such a cross-sectional view, in which the container body includes an electromagnet operatively connected to a receiver of the container body.

[0033] Figure 29 yes Figure 28 The container body is combined with a sealing member to form a partial view of a chamber, wherein a support is connected to the top plate of the chamber and cells are located on the bottom plate of the chamber. The cells are ferromagnetic and the electromagnet is turned off.

[0034] Figure 30 yes Figure 29 Another partial view of the container body, sealing components, support structure, and cells, drawn with the electromagnet activated so that the ferromagnetically labeled cells are drawn into the support structure by magnetic force.

[0035] Figure 31This is a top view of an exemplary rack containing a linearly arranged group of containers held as strips during scaffold formation and organoid formation and culture, wherein each container includes... Figure 19 The container body corresponding to the embodiment.

[0036] Figure 32 yes Figure 31 The assembly of the frame has the same Figure 19 The top view of a set of container bodies corresponding to the embodiment to form a strip.

[0037] Figure 33 yes Figure 32 Side view of the stripe.

[0038] Figure 34 It is to maintain and Figure 19 A top view of another exemplary shelf, a two-dimensional array of container bodies corresponding to the embodiment to form strips.

[0039] Figure 35 It is used for Figure 1 and Figure 2 Another embodiment of the container body is roughly as follows: Figure 25 (For related embodiments) such a cross-sectional view, wherein the bottom opening of the container body is sealed by a sealing member to form a chamber, wherein the container body defines an inlet tube, the bottom end of which is closed by a breakable barrier, and wherein a magnet is inserted into the inlet tube to place the working end of the magnet near (and above) the breakable barrier.

[0040] Figure 36 yes Figure 35 A cross-sectional view of the container body and sealing components, taken with the organoid present in the chamber and the magnet replaced by a needle with a tip that pierces the ruptureable barrier and enters the organoid.

[0041] Figure 37 yes Figure 35 A cross-sectional view of the container body and sealing components, taken with the organoid present in the chamber and the magnet replaced by a sensor / electrode having a piercing, breakable barrier and access to the sensing / stimulating end region of the organoid.

[0042] Figures 38-40 yes Figure 35 A partial cross-sectional view of the container body, taken around the ruptureable barrier before, during, and after the tip of the rupture device passes through the barrier and enters the chamber from the inlet tube.

[0043] Figure 41 yes Figure 35 The container body and sealing components are roughly as follows Figure 24(For related embodiments) such a cross-sectional view is taken so that three inlet tubes are visible, wherein the rupture device is disposed in the three inlet tubes, and only one of the rupture devices extends into the chamber below the inlet tube.

[0044] Figures 42-44 yes Figure 35 Partial cross-sectional views of a variant embodiment of the container body, wherein the fracturing barrier is configured to tear at the vulnerable mesh, wherein these views are taken around the fracturing barrier before, during and after the tip of the fracturing device passes through the fracturing barrier from the inlet tube.

[0045] Figure 45 yes Figure 35 A cross-sectional view of the container body and sealing components, taken with the organoid present in the chamber and the magnet replaced by an attenuated total internal reflection (ATR) fiber optic probe having a tip that penetrates the ruptureable barrier and enters the organoid.

[0046] Figure 46 yes Figure 35 A cross-sectional view of the container body and sealing components, taken with the organoid present in the chamber and the magnet replaced by an imaging fiber optic probe with a front lens that passes through the breakable barrier and enters the organoid.

[0047] Figure 47 yes Figure 35 A cross-sectional view of the container body and sealing components, taken with the organoid present in the chamber and the magnet replaced by an illumination probe having a distal end that enters the organoid.

[0048] Figure 48 yes Figure 35 A cross-sectional view of the container body and sealing components, taken with the organoid present in the chamber and the magnet replaced by a pneumatic device having an inflatable sac located inside the organoid to generate internal mechanical strain.

[0049] Figure 49 yes Figure 35 The container body and sealing components, such as Figure 24 (For related embodiments) such a cross-sectional view, wherein the organoid exists in a chamber and the magnet is replaced by a pair of pneumatic devices having an inflatable sac located outside the organoid to generate external mechanical strain.

[0050] Figure 50 and Figure 51 yes Figure 35 The container body and sealing components are roughly as follows: Figure 25 and Figure 24(For similar embodiments) such a truncated view, in which a pair of magnets extend into the chamber via corresponding access tubes and a prefabricated scaffold structure or biochip is mounted onto the magnets via magnetic attraction.

[0051] Figure 52 and Figure 53 yes Figure 35 A cross-sectional view of the container body and sealing components, taken in the following case: a lid is mounted on top of the container body and seals the top of the container body, and a flexible component is provided that deforms by alternating external pressure applied to a pair of reservoirs to drive liquid through the chambers alternately in opposite directions. Detailed Implementation

[0052] This disclosure provides a system for culturing, monitoring, and / or analyzing organoids or other organized multicellular structures, including methods and apparatus. Other organized multicellular structures include developing or fully developed multicellular organisms, tissue biopsies, and / or primary patient material. In an exemplary method of organoid culture, the method may include disposing a scaffold in a receiver having an open side. A sealing member may be incorporated into the open side of the receiver to form a chamber. The scaffold may be used to form the organoid within the chamber. Fluid and / or at least one substance may be introduced from an overlying reservoir into the chamber to contact the organoid.

[0053] This disclosure describes containers for forming, culturing, monitoring, and / or analyzing organoids or other organized multicellular structures, such as developing or fully developed multicellular organisms, tissue biopsies, and / or primary patient materials. The containers may be consumable (i.e., disposable after a single use) and / or may have a standard shape. The container may include a container body defining a receiver with an open side. A sealing member may be attached (e.g., coupled) to the container body to form a chamber by the receiver. The sealing member may be pre-formed or formed within the receiver, etc.

[0054] A three-dimensional (3D) structure (i.e., at least one matrix, which may be a scaffold) can be placed in the receiver before the open side of the receiver is sealed. The 3D structure can be formed in the receiver using a 3D printer. The 3D printer can dispense one or more solidifiable bio-inks, which can be mixed with cells before or during the deposition of the ink(s) to generate the 3D structure. Alternatively, the cells and the 3D structure can be introduced separately into the receiver or chamber. In other embodiments, the 3D structure can be formed at least partially or completely outside the receiver and then placed in the receiver.

[0055] When forming a stromal scaffold or after it has been formed, suitable cells that can be introduced into the stromal scaffold may include undifferentiated stem cells, differentiated stem cells that will continue to differentiate, cell aggregates, small organoids, etc.

[0056] 3D printers can also be used to apply adhesives and / or sealing fluids to the container body to allow the open side of the container to be sealed with a transparent sealing member at the end of the printing process. Exemplary printing techniques that can be applied to dispensing matrix components include droplet-based bioprinting using bio-inks, or laser-based bioprinting (e.g., laser-based direct writing of printed cells, enzymes, etc. using laser-induced forward transfer (LIFT) with pulsed lasers).

[0057] The scaffold and / or other matrix may be provided by one or more hydrogels. Each hydrogel may include one or more thermoplastic structural components, such as matrix gum, alginate, nanofibrillar cellulose, collagen, fibrin and / or polyethylene glycol, which synergistically form the matrix in a temperature-dependent manner.

[0058] In some embodiments, two or more different hydrogels / matrices may be disposed in the receiver. The hydrogels / matrices may vary depending on any suitable parameters, such as melting temperature, resistance to enzymatic degradation, solubility, cell attraction and / or cell repulsion properties, etc.

[0059] Each hydrogel / matrix may include any suitable component. Exemplary components include one or more polysaccharides (e.g., glycosaminoglycans (GAGs, such as chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, hyaluronic acid, keratin sulfate, etc.), proteoglycans (e.g., GAGs linked to a core protein (e.g., via its serine) to form substances such as aggregate proteoglycans, aggregate proteins, short-fibrillated proteoglycans, type XVIII collagen, dandruff proteins, neuroproteoglycans, basement membrane proteoglycans, leucine-rich small proteoglycans, multifunctional proteoglycans, etc.), fibrous proteins (e.g., collagen, elastin, fibronectin, laminin, etc.) and / or the like. Protease recognition sites (e.g., for scaffold metalloproteinases (MMPs)) may be bound to the hydrogel / matrix to allow for degradation / remodeling by cells. The frequency of these sites and the order of each site can be selected to allow for appropriate amounts of degradation / remodeling.

[0060] One or more growth factors may be included in the matrix during matrix formation or introduced into the liquid culture medium after its formation. Suitable exemplary growth factors include angiopoietin, bone morphogenetic protein (BMP), ciliary neurotrophic factor, community-stimulating factor, hepatic glycosides, epidermal growth factor, erythropoietin, fibroblast growth factor, glial-derived neurotrophic factor, hepatocyte growth factor, insulin, insulin-like growth factor, interleukin, leukemia inhibitory factor, keratinocyte growth factor, neuromodulatory proteins, neurotrophic proteins, platelet-derived growth factors, transforming growth factor, tumor necrosis factor (α), container endothelial growth factor, etc.

[0061] Any suitable cells can initially proliferate on the scaffold. These cells may include stem cells (e.g., pluripotent stem cells), supporting cells, etc. Cells can be deposited in the scaffold using any suitable technique, including bio-ink droplet printing, microcontact printing, photolithography, dip pen nanolithography, and / or pipetting, etc.

[0062] The container may contain multiple reservoirs in fluid communication with a chamber via channels formed in a common wall between the reservoirs and the chamber. This configuration can be described as a standard feed interface. In some embodiments, 3D printing provides a connection between the standard feed interface inside the container and any suitable printed structure, enabling the growth of different types of organoids.

[0063] The printed 3D structures can provide temporary scaffolds for appropriate types of cells as they develop into organoids. These cells can self-organize and generate their own extracellular matrix, which can replace some or all of the scaffold. The same applies to internal feeding: the container can provide a universal interface that can be optionally modified via 3D printing, and the cells can organize to optimally utilize this modified interface.

[0064] While the container is inverted, a scaffold (with or without cells) can be placed in a receiver within the container body, and a chamber can be formed through the receiver. Once these processes are complete, the container can be turned face up (to its organoid culture orientation), and at least one reservoir placed on the chamber can be filled with feed liquid. If there are no cells yet in the scaffold, suitable cells can be placed in the feed liquid, and these suitable cells can be introduced into the scaffold along with the feed liquid from the overlying reservoir (or cells can be introduced via an inlet tube (e.g., see Example 7)).

[0065] Organoid formation may require an initial culture period before a specific feeding protocol can be initiated. The feeding protocol may include loading a reservoir with appropriate culture medium and removing the medium from the reservoir according to a predetermined plan and / or based on the developmental stage or condition of the organoid. The feeding protocol may vary depending on the shape of the scaffold and the type of organoid to be formed.

[0066] The container can be configured to enable organoid monitoring via a bottom window of the chamber, which can be provided by a sealing member. In some embodiments, organoids can be monitored while they are still inside the incubator. Therefore, the incubator may include an imaging system for organoid monitoring.

[0067] The container enables light sheet 3D imaging. The container's chamber may have two, three, or more optical windows, and light can propagate into and / or out of the chamber through each window. For example, the container may have a bottom window, which may all be planar, and one or more transverse windows. In some embodiments, the container may have a pair of transverse windows arranged opposite each other.

[0068] Multiple container bodies (and containers) can be organized into strips. These strips can be formed during manufacturing by pre-connecting the container bodies to each other in a linear or two-dimensional array (e.g., by integrally joining or shaping the container bodies together, such as by injection molding). Alternatively, the strips can be formed during manufacturing or by the user assembling the individual container bodies with suitable strip holders. In some embodiments, the strip holder can be configured to hold only one or two strips to allow space for imaging objectives. The strips of the container bodies can be loaded into a 3D printer to allow the printer to print supports in each container body and / or add sealing members to each container body.

[0069] This disclosure enables the production of large functional organoids by feeding them with different internal and external culture media. The average or maximum diameter of the large organoids can be greater than about 0.1, 0.2, 0.5, 1, or 2 mm, etc. Research on large organoids remains challenging, with researchers facing two main limitations. First, each type of organoid requires different culture conditions, such as specific hydrogels, substrates, or even mechanical properties (e.g., shear forces caused by culture medium flow). Second, microscopy of large organoids is extremely challenging. Existing methods still involve thin sectioning and staining of organoid materials, and image acquisition of fixed samples using confocal scanning microscopy or even slide readers.

[0070] This disclosure provides a system and method that helps overcome one or two obstacles. By using a combination of 3D printing (scaffolds and / or cells) and culture medium exchange via gravity flow, users can generate unique 3D environments optimized for each type of organoid. Various different types of organoids can be grown. Feeding and waste removal can be addressed through fluid communication between the container's chamber and the overlying reservoir. Integrating optical windows into each container, with at least one window for excitation of light entry and another for emission of light exit, allows for monitoring of the organoid's live cells via light sheet microscopy. Alternatively or additionally, the organoid can be imaged via one or more windows using classic wide-field microscopy. Therefore, the containers disclosed herein enable live-cell microscopy of developing and / or mature organoids. High-volume and / or high-throughput microscopy can be performed on the organoids.

[0071] Other aspects of this disclosure are described in the following sections:

[0072] (I) A container for organoid formation, culture, monitoring and / or analysis, (II) A method for organoid formation, culture, monitoring and / or analysis, and / or (III) Examples.

[0073] I. Vessels for organoid formation, culture, monitoring and / or analysis

[0074] This section describes an exemplary container 50 for the formation, culture, monitoring, and / or analysis of organoids 52 (or other organized multicellular structures); see also Figure 1 and Figure 2 Container 50 is schematically shown here; the front and rear walls of the container are not visible to distinguish the closure of the container. Figure 1 ) or open ( Figure 2 The top and bottom sides of ).

[0075] Figure 1 A container 50 is shown containing an organoid 52 within a chamber 54, wherein the organoid is immersed in a culture medium 56 (e.g., a liquid or semi-solid culture medium to promote organoid growth and development). The organoid 52 may be attached to an upper region of the chamber 54, such as the top wall 58 of the chamber (the top wall is interchangeably referred to as the top plate). The transverse walls 60 and the bottom wall 62 of the chamber may provide barriers to impede fluid passage, thereby fluid-sealing all chambers below the top wall 58.

[0076] Container 50 defines a plurality of compartments 63 located above chamber 54. Each compartment 63 may share a wall with chamber 54 and may be initially in fluid communication with or not initially in fluid communication with chamber 54. An exemplary compartment includes a reservoir and an inlet tube (see Example 7). Here, container 50 includes (at least) two reservoirs 64a, 64b for receiving respective liquid culture media 66a, 66b above chamber 54. Each reservoir 64a, 64b may be in fluid communication with chamber 54 independently of the other reservoirs via a corresponding dedicated channel 68a, 68b. Each channel 68a, 68b may extend from one of the reservoirs 64a, 64b through top wall 58 to chamber 54 and may be flush with the top and bottom sides of top wall 58, or each channel 68a, 68b may protrude from the top and / or bottom sides of top wall 58 as an annular protrusion (e.g., see Example 4). In other examples, container 50 may have at least, exactly 3, 4, 5, 6 or more overlying reservoirs, each of which is in fluid communication with chamber 54 via top wall 58 and / or in fluid communication with chamber 54 independently of other reservoirs (see, for example, Example 3).

[0077] Two or more reservoirs of container 50 may contain any suitable substance to be supplied to chamber 54. Exemplary substances include nutrients, effectors, and reagents. Suitable nutrients include any substance that promotes cell health and proliferation within chamber 54 and thus promotes the growth and development of organoids 52. Exemplary nutrients may include sugars (such as glucose), amino acids, proteins, nucleotides, vitamins, minerals, fatty acids, etc. Effectors include any molecule (such as an inducer or inhibitor) that activates, controls, or deactivates processes or effects (such as differentiation, protein synthesis, migration, etc.). Exemplary effectors include anticancer compounds, growth factors, differentiation factors, oligonucleotides, mRNA, etc. Reagents include any compound that facilitates organoid analysis. Exemplary reagents include markers, fixatives, and scavengers, etc. Markers may include dyes (e.g., visible colorants and / or photoluminescent dyes). A photoluminescent dye is any substance that emits light in response to excitation light.

[0078] Each reservoir 64a, 64b may have a transverse wall 70 to laterally accommodate fluid. For example... Figure 1 As shown, at least one transverse wall may be shared between at least a pair of adjacent reservoirs. In other examples, at least a pair of reservoirs of the same or different containers may be in transverse fluid communication with each other, such as via a channel formed in the wall 70 shared between the reservoirs, regardless of the chamber 54 (see, for example, Example 2).

[0079] Each reservoir 64a, 64b may have an open top 72 to facilitate the introduction and removal of fluid using a fluid transfer device (e.g., a pipette or other pump). Container 50 may include a single removable cap 74 that engages with the container to cover the open top 72 of each reservoir 64a, 64b during incubation in an incubator. Alternatively, the container may include two or more caps that can be removed independently of each other and collectively cover all reservoirs. Each cap 74 may have a flange 76 configured to vertically overlap the upper end of each reservoir and restrict lateral movement of the cap when covering the reservoir, optionally without creating a tight fit. In some embodiments, the cap may be a cap-shaped element that forms a fluid-tight seal on top of one or more reservoirs (e.g., see Example 8).

[0080] Figure 2 It shows Figure 1 An exploded view of container 50, wherein the container is empty. Container 50 may include a lid 74, a container body 78, and a sealing member 80. Container body 78 may define reservoirs 64a, 64b and a receiver 82 below the reservoirs. Receiver 82 provides a top wall 58 and a transverse wall 60 for chamber 54. However, receiver 82 may have an open bottom side 84, which can be sealed with a flat sealing member 80 to form chamber 54 after the support scaffold 86 supporting the organoid formation has been arranged in receiver 82 (see [reference]). Figure 1 (See Section II). A sealing member may be attached to the bottom end of the receiver 82 (and / or container body 78) to form a chamber 54. In other embodiments, the sealing member may be formed in situ to seal the bottom side 84 of the receiver 82 (see Section II).

[0081] Container 50 may have one or more optical windows to facilitate imaging (see...). Figure 1 Each optical window can be used to propagate light into the chamber to illuminate at least a portion of the chamber 54, or to receive light from the chamber, such as for imaging. In an exemplary embodiment, the container 50 has a bottom window 88 provided by the sealing member 80, and one or more transverse windows 90 provided by the container body 78. Each optical window can be transparent and / or planar. The bottom window 88 can be transverse to (e.g., orthogonal to) each transverse window 90, and the transverse windows can be formed by opposing transverse walls 60 of the chamber 54. (As used herein, the term "light" refers to optical radiation, including ultraviolet radiation, visible light radiation (i.e., visible light), and / or infrared radiation.)

[0082] The components of container 50 can be formed from any suitable material using any suitable process. In an exemplary embodiment, the container body 78 may be formed from a polymer (such as a transparent polymer). The container body may not have removable / movable parts and / or may be formed as a single piece, such as by injection molding, such that all structures of the container body (e.g., compartments) are integrally formed with each other. Thus, the receiver 82 (and / or chamber 54) and reservoirs 64a, 64b may have fixed positions relative to each other and / or may be non-removably / securely attached to each other. The sealing member 80 may be formed from glass or a polymer, and may be pre-formed, at least partially or completely, inside the receiver 82 or formed in situ.

[0083] The chamber 54 (and / or receiver 82) and each reservoir 64a, 64b can have any suitable size. The chamber 54 can have a volume of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, or 1 mL. The chamber size can be designed to accommodate organoids of any suitable size (e.g., the largest size), such as organoids having a diameter of at least about 0.2, 0.5, 1, 2, 3, 4, or 5 mm, etc. In an exemplary embodiment, each overlying reservoir of the container is at least as large as, or about 2, 5, or 10 times the volume of the chamber 54 (and / or receiver 82), such as having a volume of at least about 0.5, 1, 2, 4, or 6 mL, etc.

[0084] Container 50 and / or any of its compartments can have any suitable geometry. For example, each of the receiver 82, chamber 54, and / or each reservoir (e.g., reservoirs 64a, 64b) can independently have a polygonal (e.g., rectangular), elliptical (e.g., circular), oval, rosette-shaped, or other shaped cross-section (e.g., in a horizontal plane). Thus, receiver 82, chamber 54, and / or each reservoir can independently be cylindrical, truncated conical, rectangular prism, conical prism, or combinations thereof.

[0085] Other aspects of the container 50, which can be used for organoid formation, culture, monitoring and / or analysis, are described below.

[0086] II. Methods for organoid formation, culture, monitoring and / or analysis

[0087] This section describes methods for forming, culturing, monitoring, and / or analyzing organoids (or other organized multicellular structures) in the containers disclosed herein; see also Figures 3-15 The method steps described in this section can be performed using any container, container feature, and / or procedure described elsewhere in this document, in any suitable order and combination.

[0088] Figures 3-10Exemplary configurations of containers 50, organoids 52, and culture media 56, 66a, 66b produced by implementing organoid culture and analysis methods are shown. At least one container body 78 of at least one container 50 may be used. In some embodiments, the container bodies 78 may be selected as linear or a two-dimensional array (e.g., see Examples 1 and 6), or an array may be created after at least a subset of the following steps have been performed.

[0089] Figure 3 The container body 78, selected and placed in an inverted orientation, is shown, with the receiver 82 opening at the top. In other words, the top wall 58 is located below the bottom side 84 of the opening. This orientation utilizes gravity to facilitate fluid delivery to the top wall 58, while the transverse wall 60 prevents fluid from flowing laterally out of the receiver 82.

[0090] The scaffold 86 for organoid formation can be disposed within the receiver 82. For example... Figure 4 and Figure 5 As shown, the scaffold may be contained within at least partially in-situ generated hydrogel 92. For example, the scaffold 86 of the hydrogel 92 may be 3D printed onto the top wall 58 by a 3D printer 94. The 3D printer 94 may utilize any suitable technology to deliver the components of the scaffold. In some embodiments, the 3D printer may utilize inkjet technology to deliver fluid droplets (such as droplets 95) that may contain scaffold structural components, cells 96, tubes, cage-like effectors, growth factors, etc. Alternatively or additionally, the 3D printer 94 may utilize laser-induced forward transfer to deliver components (e.g., cells 96), etc. In other embodiments, at least a portion of the scaffold 86 may be formed separately from the container body 78 and subsequently placed into the receiver 82 (e.g., attached to the top wall 58).

[0091] Hydrogel 92 may be a thermotropic reversible gel or may not be a thermotropic reversible gel. The hydrogel may have a gel point (gelation temperature) higher than the organoid culture temperature, allowing the hydrogel scaffold to form by cooling during scaffold printing. Alternatively or additionally, the polymerization reaction that forms the hydrogel may be photoinduced using optical radiation such as ultraviolet or visible light.

[0092] The scaffold 86 (and / or hydrogel 92) may horizontally overlap the bottom ends of one or more channels 68a, 68b. For example, in the depicted embodiment, a through-axis (e.g., a vertical axis) defined by channel 68b intersects with hydrogel 92 and extends through the scaffold 86. In other embodiments, the scaffold 86 (and / or hydrogel 92) may overlap with multiple channels 68a, 68b.

[0093] During the formation of the scaffold 86 in the hydrogel 92, one or more channels 68a, 68b can be extended by 3D printing to facilitate the supply of nutrients and / or effectors to the hydrogel 92 and the cells 96 therein during organoid formation and culture. The resulting channel extensions can be one or more laterally permeable tubes embedded in the hydrogel 92 and can branch to form a channel network within the hydrogel. The channel network and / or the laterally permeable tubes embedded in the hydrogel 92 can extend between at least one pair of channels 68a, 68b of the container body 78. Effectors (e.g., differentiation factors) can be supplied via the channel network to establish a concentration gradient within the hydrogel for more controlled stem cell differentiation and organoid formation. These tubes can be formed by photoinduced polymerization or thermal polymerization, etc. Pre-formed tubes can also, or alternatively, be incorporated into the hydrogel or scaffold to further promote fluid flow within the hydrogel / scaffold.

[0094] The opening side of the receiver 82 can be hermetically sealed to form a chamber 54 containing the hydrogel 92 and the support 86. For example... Figure 6 As shown, a sealing member 80 can be attached to the end of the receiver 82. Alternatively, as described below, the sealing member 80 can be generated at least partially in situ within the receiver 82 by polymerization and / or curing of a sealing fluid. The receiver 82 can be sealed before or after the cells 96 have been placed into the receiver, and / or before or after the culture medium 56 has entered the receiver 82 (and / or chamber 54) around the hydrogel 92.

[0095] See Figure 7 Culture media 56, 66a, 66b can be placed in chamber 54 of container 50 and in each reservoir 64a, 64b. The same culture medium can be introduced into each chamber and reservoir, or different culture media can be introduced, which can create a concentration gradient of one or more nutrients and / or effectors. As described above, culture medium 56 can initially be introduced via the open side of receiver 82 before the receiver is sealed to form chamber 54. To prevent leakage of culture medium 56 from receiver 82 through one or more channels 68a, 68b before chamber 54 is formed, the end of each channel can be covered with hydrogel 92 (or different hydrogels). Different hydrogels can be configured to melt below the culture temperature (e.g., 37°C) for organoid formation and culture. Alternatively, culture medium 56 can be introduced into chamber 54 from at least one reservoir 64a, 64b via at least one channel 68a, 68b (after chamber formation).

[0096] If the fluid levels in the reservoirs are different, gravity can drive fluid from one of the reservoirs into chamber 54, and / or drive fluid between the reservoirs via the chambers. For example, in Figure 7In container 50, the top of culture medium 66a in container 64a is higher than the top of culture medium 66b in container 64b. Therefore, gravity drives culture medium 66a into chamber 54 via channel 68a (which serves as an inlet) and out of chamber 54 via channel 68b (which serves as an outlet). Over time, the levels of culture media 66a and 66b will tend to be equal. Therefore, to drive further flow of fluid in the same or opposite directions, fluid can be added to or removed from one or more containers, or the vertical orientation of container 50 can be periodically changed (e.g., see Example 2).

[0097] Figure 8 and Figure 9 A container 50 containing organoids 52 is shown. The organoids are formed through the proliferation and differentiation of cells 96 within the hydrogel 92. Figure 9 In the process, as scaffold 86 is remodeled by organoid cells, the size and shape of organoid 52 relative to... Figure 8 This has been changed. In other embodiments, the final size and shape of the organoid 52 may be substantially defined by the size and shape of the scaffold 86 at the time of formation.

[0098] Figure 10 An exemplary imaging system 100 is shown for capturing an image 102 of an organoid 52 using light sheet microscopy. The imaging system may include an illumination assembly 104, which includes a light source 106 for generating light for a light sheet 108. Figure 10 As shown, the light sheet 108 can be horizontally oriented and can propagate through the organoid 52 from one of the transverse windows 90. Alternatively, the light sheet 108 can be vertically oriented and can propagate vertically through the organoid 52 from the bottom window 88. Light can be collected from the organoid 52 using the objective lens 110 for capture by the image sensor 112. Light propagating through the bottom window 88 can be collected, as in the depicted embodiment, or light can be collected from one of the transverse windows 90. The light sheet 108 can be moved by corresponding movement of the illumination assembly 104 (or container 50) to allow stacking of captured images, thereby providing three-dimensional image data of the organoid. If the organoid 52 no longer needs to be viable, fixation and removal reagents can be provided from one or more reservoirs 64a, 64b through the top wall 58 of the chamber 54 to fix and remove the organoid prior to imaging.

[0099] Figures 11-13 An alternative in-situ method for sealing the open side 84 of the receiver 82 to form the chamber 54 is shown. Figure 11An inverted container body 78 is shown after the formation of a hydrogel 92 including a scaffold 86. A filler hydrogel 113 has also been dispensed into the open receiver 82 via the open side 84, which prevents leakage from the receiver 82 through the channel 68a. The filler hydrogel can be configured to dissolve or thaw once organoid culture begins. Figure 12 A 3D printer 94 is shown dispensing sealing liquid 114 into a receiver 82. Figure 13 The solidification of sealing liquid 114 is shown to airtightly seal the open side of receiver 82, thereby forming chamber 54.

[0100] The sealing liquid can be configured to cure to produce a thermoplastic polymer or a thermosetting polymer, etc. Exemplary thermoplastic polymers include thermoplastic elastomers or waxes, preferably having a melting point below 100°C. A lower melting point is desirable, but at least about 50°C. The thermoplastic polymer can be dispensed in liquid form onto the surface of the filler hydrogel 113 at a temperature above the polymer's melting temperature to form a sealing layer 116, which hardens as the layer cools, thereby producing a sealing member 80.

[0101] In other examples, the layer can be curable to produce a thermosetting polymer. Figure 13 An example is shown in which the sealing layer 116 is cured into a thermosetting polymer by irradiation with light radiation (e.g., ultraviolet light) from the illumination assembly 104. The illumination assembly forms a light sheet 108 that preferentially irradiates the sealing layer 116 with ultraviolet light, thereby minimizing light damage to the scaffold 86 and / or cells 96 already present in the receiver 82.

[0102] Figure 14 The image shows the container body 78 flipped to its organoid culture orientation, and culture media 56, 66a, 66b in chambers 54 and reservoirs 64a, 64b, wherein a sealing layer 116 is solidified to form a sealing member 80. An objective lens 110 of the imaging system collects light from chamber 54 to form an image. The culture media 56 and the sealing layer 116 may have substantially the same refractive index to improve image quality.

[0103] Figure 15 An improved in-situ method (with) is shown for the open side of the receiver 82 for sealing the container body 78. Figure 13 (Compared to) Before the thermosetting sealing liquid layer 116 is added to the receiver 82, a radiation-blocking layer 118 can be formed on the surface of the filler hydrogel 113. Layer 116 can be irradiated with light radiation from a light source 120 located above the container body 78 to promote curing of layer 116, thereby forming the sealing member 80. The radiation-blocking layer 118 protects the hydrogel 92 from light damage.

[0104] III.Examples

[0105] This section describes other embodiments of systems and methods for organoid formation, culture, monitoring, and / or analysis. These embodiments are for illustrative purposes only and should not limit the entire scope of this disclosure.

[0106] Example 1. Bands in organoid culture

[0107] See Figure 16 This example describes an exemplary strip 130 for forming, culturing, monitoring and / or analyzing organoid arrays.

[0108] Strip 130 may include an array of container bodies 78 connected to each other. The container bodies may be integrally formed with each other, or formed independently and then connected to each other after formation (and / or after the receiver 82 seals the formed chamber 54) (see also...). Figure 1 The independently formed container bodies 78 can be connected to each other by bonding, interference fit, fasteners or retainers (see, for example, Example 6). The container bodies can be connected to each other to form a container 82 in a linear or at least two-dimensional array.

[0109] Example 2. Fluid transfer between containers

[0110] This example describes an exemplary configuration for transferring fluid between reservoirs 64a, 64b of the same or different containers 50; see also Figure 17 , Figure 17A , Figure 17B and Figure 18 .

[0111] Figure 17 A strip 130 is shown during the culture of organoids 52 in chamber 54. Each container 50 has an independent flow cycle indicated by arrows 132, 134. A flow 132, driven by gravity, passes through each chamber 54 between channels 68b and 68a due to the level difference of culture media 66a, 66b in the respective reservoirs 64a, 64b. A supplemental flow 134, driven by pump 136, flows from reservoir 64a to reservoir 64b. The flow rate of pump 136 can be adjusted to maintain the respective levels of culture media 66a, 66b substantially constant, such that fluid flows through chamber 54 at a substantially constant rate. This flow can apply pressure to the organoids 52, which can promote organoid growth and development. In other examples, the flow rate of pump 136 can be varied, thereby applying varying pressure to the organoids 52.

[0112] In other embodiments, pump 136 can be removed, such as... Figure 17A and Figure 17BThe container strip 130 is shown in the diagram. The strip 130 can be rocked to tilt the retainer at an appropriate rate from the vertical direction, so that the direction of the gravity-driven flow 132 is periodically reversed. In other words, the retainer can be tilted in one direction to drive fluid from reservoir 64a to reservoir 64b within each container 50 via channels 68a, 68b, and then tilted in the opposite direction of rotation to drive fluid from reservoir 64b to reservoir 64a within each container 50 via channels 68a, 68b.

[0113] Figure 18 Another band 140 is shown during the culture of organoid 52 in chamber 54. Band 140 is similar to... Figure 16 and Figure 17 The strip 130 drives a flow 132 between channels 68a, 68b in each container 50. However, corresponding channels 142 provide fluid communication between each pair of adjacent containers 50. More specifically, as indicated by flow arrow 144, each channel 142 allows fluid to flow directly from a reservoir 64a of one container 50 to a reservoir 64b of an adjacent container 50, and vice versa. To maintain fluid circulation, the strip 140 can be periodically tilted (e.g., Figure 18 (As shown) the flow of fluid is directed from left to right toward one end of the holder, and then tilted in the opposite direction of rotation to direct fluid flow from right to left toward the opposite end of the holder. Pump 146 can transfer culture medium between reservoirs 64a, 64b located at opposite ends of the strip 140, as indicated by the flow arrow at 148. The pump can be used in place of periodically tilting the strip 140, or in addition to periodically tilting the strip 140.

[0114] Example 3. Container Examples

[0115] This example describes an exemplary embodiment 150 of container 50 in Chapter I, which has a container body 78 formed by injection molding and defines four reservoirs 64a to 64d; see also Figures 19-25 .

[0116] Each reservoir 64a to 64d may be in fluid communication with the receiver 82 (and / or chamber 54) via a corresponding channel 68a to 68d extending through the top wall 58 of the receiver 82 (and / or chamber 54) (see [link]). Figure 23 and Figure 25 The top of each channel can be flush with the top wall 58 (see...). Figure 25 ).

[0117] Various optical windows can be incorporated into the container 150. The sealing member is already attached to the bottom surface 152 of the container body 78 (see...). Figure 21Subsequently, a planar sealing member 80 (e.g., similar to a microscope coverslip) can provide a bottom window 88 for chamber 54. The container body may include a pair of transverse windows 90 (see...). Figures 19-21 The pair of horizontal windows can be slightly tilted from the vertical direction to facilitate manufacturing by injection molding (see...). Figure 24 ).

[0118] Example 4. Container with tube

[0119] This example describes an exemplary container with pre-formed and / or in-situ formed tubes; see also Figure 26 and Figure 27 .

[0120] Figure 26 Another embodiment 160 of container 50 is shown, for use with Figure 25 Compare with container 150. Channels 68a to 68d of container 160 differ from those of container 150. More specifically, channels 68b and 68c are partially defined by annular protrusions 162 projecting from the underside of the top wall 58 of receiver 82 (and / or chamber 54). The lumen of any or all channels may be formed at least partially by the annular protrusions.

[0121] Figure 27 A transversely porous tubular extension 164 is shown, which can be formed in situ on one or more annular protrusions 162 by 3D printing. Each tubular extension may be branched or unbranched. In the depicted embodiment, the tubular extension 164 connects channels 68b and 68c to each other. In other embodiments, a porous tubular network can be formed that connects any suitable subset or all of the channels to each other. Each tubular extension 164 may be supported by and / or embedded in hydrogel 92 (see Section II). The tubular extension may have the same composition as the hydrogel or may have a different composition to, for example, minimize / promote cell attachment and / or remodeling.

[0122] Example 5. Container with electromagnet

[0123] This example describes an exemplary embodiment 170 of a container 50 having an electromagnet 172 positioned to attract ferromagnetically labeled cells 174 when the electromagnet is energized (i.e., turned on); see also Figures 28-30 .

[0124] Container 170 may be constructed similarly to container 150 and may have any suitable combination of the container features disclosed herein (see [link to document]). Figure 28The electromagnet 172 may have a working end 176, which is very close to or located within the receiver 82 and / or chamber 54, near at least one of channels 64a to 64d. In the depicted embodiment, the working end 176 is located centrally between channels 64b and 64c. The electromagnet may be energized via its upper end 178.

[0125] Figure 29 and Figure 30 This illustrates how an electromagnet 172 can be used to attract ferromagnetically labeled cells 174 into a hydrogel 92 and / or scaffold 86 within a chamber 54. The scaffold 86 and hydrogel 92 can be formed as described above. Ferromagnically labeled cells 174, along with culture medium 56, can be introduced into the receiver from one or more overlying reservoirs via one or more channels 68a to 68d before or after the receiver is sealed to form chamber 54. In any case, as Figure 29 As shown, the ferromagnetically labeled cells 174 are initially on the outside of the hydrogel 92 and can settle into the bottom chamber 54 before the electromagnet is turned on. Figure 30 The diagram illustrates how energizing an electromagnet attracts ferromagnetically labeled cells 174 to and / or into scaffold 86. Before introducing the cells into the receptor / chamber, the ferromagnetically labeled cells can be combined with a ferromagnetic material (e.g., a compound containing iron, cobalt, and / or nickel). For example, ferromagnetic particles (such as iron oxide particles) can be provided to the cells to make them ferromagnetic, or the cells can be coated with a ferromagnetic probe, and so on.

[0126] Example 6. Shelves for container modules

[0127] This example describes exemplary racks 180 and 182 that hold containers 150 in a linear array or a two-dimensional array to form strips 130, respectively; see also Figures 31-34 .

[0128] Each shelf 180, 182 (interchangeably referred to as a retainer) has a series of openings 184 to receive the container body 78 of the container 150. The openings 184 may be arranged along a single line (shelf 180) or in a rectangular grid pattern (shelf 182), etc. Each container body 78 may be detachably placed in the opening 184. The size and shape of the openings may be designed to prevent the container body from passing completely through the opening. The container body may be attached to the opening 184 by any suitable mechanism, including snap-fit ​​or individual retainers, etc.

[0129] Example 7. Container with inlet tube

[0130] This example describes an exemplary container including an inlet tube, and the use of the inlet tube for housing various instruments; see also Figures 35-51 .

[0131] Figure 35 An embodiment 190 of a container 50 is shown, comprising a container body 78, the container body 78 being sealed at its bottom end with a sealing member 80 to form a chamber 54. Except that the container body defines at least one inlet tube 192, the container body 78 is similar to the container body described above for container 160 (see [link to previous description]). Figure 26 The inlet tube opens at its top end 194, but may have a bottom end closed by a barrier 196. This barrier may be a wall region shared by the top wall 58 of the chamber between the inlet tube 192 and the chamber 54, and this wall region prevents fluid communication between the inlet tube and the chamber when the barrier is intact. For simplicity, in any of the figures of Example 7, the chamber 54 and reservoirs 64a, 64b are not shown as containing fluids (e.g., culture media or other liquids).

[0132] As further described below, barrier 196 can be configured to be ruptured by a sharp or blunt instrument to enter chamber 54. Therefore, as... Figure 35 As shown, the barrier may be thinner than the adjacent area of ​​the top wall 58, and / or the barrier may include a predetermined structure (e.g., a predetermined fragile area with a smaller thickness) at which the barrier may be preferentially torn or broken with a suitable tool.

[0133] Figure 35 An exemplary device 198 is shown inserted into the access tube 192. Here, device 198 is a permanent magnet 200 with its magnetic poles 202 adjacent to the barrier 196. In other embodiments, the magnet may be an electromagnet or any other device disclosed herein. The magnet 200 may be strong enough to create a magnetic field extending into the chamber 54 if the barrier 196 is intact. In other cases, the bottom end of the magnet 200 may be inserted into the chamber 54 after the barrier 196 has been broken (and even if the magnet can break the barrier). Regardless of whether the magnet is inserted into the chamber 54, it may exert an attractive force on ferromagnetic articles (e.g., ferromagnetically labeled cells) within the chamber 54. The magnet may be retractable / removable and / or may be securely attached to the access tube 192.

[0134] Figure 36 The container 190 is shown, in which organoids 52 are present in chamber 54, and magnet 200 is replaced by a device 198 configured as needle 204 (with...). Figure 35(Compared to). The sharp tip 206 of needle 204 has pierced barrier 196 (now referred to as ruptured barrier 196') and has entered organoid 52. The needle may be hollow or solid. In either case, the needle may allow a sample of organoid 52 to be collected and removed from chamber 54 via access tube 192 for organoid biopsy. Alternatively, or additionally, the needle may be used to introduce fluids, cells, and / or effectors, etc., into organoid 52 and chamber 54. For example, the needle may allow tissue or cells to be transplanted into the organoid to achieve xenograft, allogeneic, or allogeneic transplantation.

[0135] Figure 37 As shown Figure 36 The container 190, except for the device 198, is a sensor / electrode 208 including an electrical connector 210 (e.g., one or more conductive wires). The sensing / stimulation end region 212 of the sensor / electrode 208 has pierced the barrier 196 (now a ruptured barrier 196') and entered the organoid 52. In other embodiments, the barrier 196 may first be ruptured by a different device 198, such as a dedicated rupture device, and then replaced by the sensor / electrode 208 (or any other device disclosed herein, etc.). The sensor / electrode 208 may include at least one electrode or electrode array for electrical stimulation of the organoid 52. Alternatively, or additionally, the sensor / electrode 208 may include any suitable sensor(s), such as an electrical sensor (e.g., a capacitive sensor), a pH sensor for measuring pH, an electrochemical sensor, an oxygen sensor, a CO2 sensor, and / or the like.

[0136] Figures 38-40 The illustration shows the device 198, configured as a piercing instrument 214, before, during, and after the barrier 196 at the bottom end of the inlet tube 192 is pierced. Figure 38 In the middle, the tip 216 of the rupture device 214 travels downward (indicated by the movement arrow at 218) and almost reaches the barrier 196. Figure 39 In the middle, the tip 216 contacts the barrier 196 and applies a deformation force to the barrier. Figure 40 In this process, tip 216 has completely penetrated the barrier and entered chamber 54. The wall region forming barrier 196 can be sufficiently resilient to maintain radial contact with rupture device 214 after the barrier has been punctured. This radial contact can form a fluid tight seal 220 between container body 78 (at the ruptured barrier 216') and rupture device 214, which substantially prevents fluid in chamber 54 from traveling upward into inlet tube 192.

[0137] Container 190 may have only one inlet pipe 192 or multiple inlet pipes 192. For example, Figure 41 It shows roughly as follows Figure 24The container 190 is thus cut off, making the three inlet tubes 192 visible. As described above, each inlet tube 192 may terminate at a corresponding barrier 196. Furthermore, each barrier 196 may be a wall region of the top wall 58 located between the corresponding inlet tube 192 and the chamber 54 and / or a shared wall region between the inlet tube 192 and the chamber 54. Thus, the wall region may be a common wall region or may not be a common wall region. In the depicted embodiment, a corresponding rupture device 214 is disposed in each of the three inlet tubes 192, wherein only one rupture device extends into the chamber 54. (A ruptured barrier is indicated by 196'.) In other embodiments, any suitable number of barriers 196 may be ruptured, thereby providing access to the chamber 54 for any suitable combination of devices 198.

[0138] Figures 42-44 As shown Figures 38-40 Thus, partial cross-sectional views of different embodiments 230 of container 50 are taken before, during, and after the use of the rupture device 214 to rupture barrier 196. The barrier 196 of container 230 has a fragile mesh 232 at which the barrier is preferentially torn in response to the pressure applied by the rupture device 214. Figure 44 In this configuration, the end of the rupture device 214 has entered the chamber 54. Fluid can travel upward from the chamber 54 into the inlet tube 192, or the fluid travel can be limited by a tight radial fit between the rupture device 214 and the inlet tube 192 and / or by an airtight seal around the rupture device at the top of the inlet tube 192.

[0139] Figure 45 A container 190 is shown having an attenuated total internal reflection (ATR) fiber optic probe 234 disposed in an inlet tube 192 and extending into a chamber 54 within an organoid 52. The ATR probe 234 has an illumination fiber 236 to guide light from a light source to the bottom end of the probe 234 (as shown in 238), and the ATR probe 234 also has a sensor fiber 240 to guide light from the bottom end to an optical sensor (e.g., a spectrometer, photometer, etc.) (as shown in 242). The probe 234 may allow the use of IR spectroscopy (e.g., for chemical analysis of organoids), Raman spectroscopy (such as surface-enhanced Raman spectroscopy), etc.

[0140] Figure 46 A container 190 is shown having a fiber optic imaging probe 244 disposed in an access tube 192 and extending into a chamber 54 within an organoid 52. The imaging probe 244 may have an endoscope-mounted lens 246 and a fiber bundle 248 for propagating light from the lens 246 to an image sensor. The imaging probe may allow for endoscopic imaging, confocal imaging, and / or illumination-only imaging (e.g., for imaging using an image sensor not coupled to the fiber bundle 248, for optogenetics, etc.).

[0141] Figure 47 A container 190 is shown having an illumination device 250 disposed in an inlet tube 192 and extending into a chamber 54 within an organoid 52. The illumination device 250 may include an optical fiber 252 to direct light from a light source 254 to an outlet aperture 256 at the distal end of the optical fiber 252. Exemplary uses of the illumination device 250 include illuminating the organoid 52 for imaging by an image sensor not coupled to the optical fiber 252, as a wavefront sensing light source (adaptive optics), as a light source for optogenetics, etc.

[0142] Figure 48 A container 190 is shown having a pneumatic device 258 located in an inlet tube 192. The pneumatic device 258 has an inflatable sac 260 located within an organoid 52. This sac is operatively connected to a pressure regulating device (such as a pump 262) that can inflate the sac 260 to create internal mechanical strain within the organoid 52, as shown in 264. The pneumatic device 258 can inflate and contract the sac 260 to expand and shrink it as needed. The sac 260 may be printed together with a support 86 and subsequently connected to the tube 266 of the pneumatic device 258. In other embodiments, the sac 260 may be introduced into the organoid 52 via a needle or other sharp object.

[0143] Figure 49 As shown Figure 41 Such a container 190, but also having a pair of pneumatic devices 258a, 258b disposed in the corresponding inlet pipe 192. In addition to having an inflatable sac 260 located outside the organoid to generate external mechanical strain on opposite sides of the organoid 52, each pneumatic device is... Figure 48 It is similar to a pneumatic device.

[0144] Figure 50 and Figure 51 The following are shown respectively: Figure 35 and Figure 36 Such a container 190, but also having a pair of mounting magnets 270a, 270b extending into the chamber 54 via corresponding inlet tubes 192. Magnets 270a, 270b magnetically attract corresponding magnets 272a, 272b that have been magnetically attached to the prefabricated scaffold structure 274 or biochip.

[0145] Example 8. Containers with flexible membranes

[0146] This example describes an exemplary container 190 that utilizes one or more flexible membranes (interchangeably referred to as diaphragms) to drive fluid flow within the container; see also Figure 52 and Figure 53 .

[0147] Container 190 includes a cap-shaped member 282 mounted on top of container body 78. The cap-shaped member 282 forms an airtight seal with container body 78. The cap-shaped member includes a frame 284 that fits snugly onto the top edge of container body 78. At least one flexible membrane 286 is mounted to frame 284 and covers at least two reservoirs (e.g., 64a, 64b). The undeformed configuration of the flexible membrane is shown as a dashed line at 288. Pressure 290 can be applied to the flexible membrane 286 above reservoir 64a or 64b to push the membrane downward, as shown at 292. This pressure drives fluid to flow from one reservoir through chamber 54 to the other reservoir, as shown by arrows 294, 296. The flexible membrane 286 can responsively deflect upward, as shown at 298. Figure 52 and Figure 53 As shown, pressure can be applied to the flexible membrane 286 or above the reservoirs 64a, 64b to drive fluid in opposite directions between the reservoirs.

[0148] Example 9. Selected Examples

[0149] This example describes selected embodiments of this disclosure as a series of index paragraphs.

[0150] Paragraph A1. A method for culturing and / or analyzing organoids, the method comprising: (a) sealing the open side of a receptor to form a chamber; and (b) forming an organoid in the chamber.

[0151] Paragraph A2. According to the method described in paragraph A1, the sealing includes attaching a sealing member to the open side of the receiver.

[0152] Paragraph A3. According to the method described in paragraph A2, the attachment of the sealing member includes attaching the sealing member to the receiver.

[0153] Paragraph A4. According to the method described in paragraph A3, wherein the bonding includes bonding a prefabricated sealing member to the receiver.

[0154] Paragraph A5. The method according to paragraph A2 or A3, wherein sealing includes at least partially hardening / curing the sealing material in the receiver to form a sealing member.

[0155] Paragraph A6. The method according to paragraph A5, wherein the sealing material comprises a thermosetting resin.

[0156] Paragraph A7. The method according to paragraph A6, wherein sealing includes forming a thermosetting resin layer in the receiver and irradiating the thermosetting resin layer with electromagnetic radiation (such as ultraviolet light) to cure the thermosetting resin.

[0157] Paragraph A8. The method according to paragraph A7, wherein the receiver contains a scaffold to promote organoid formation, and wherein irradiation is performed using a light sheet positioned and oriented to preferentially irradiate the thermosetting resin layer relative to the scaffold.

[0158] Paragraph A9. The method according to paragraph A7, wherein the receiver comprises a scaffold to facilitate organoid formation, and further comprises a photoresist layer located between the thermosetting resin layer and the scaffold, and wherein irradiation is performed to allow light to propagate through the thermosetting resin layer to the photoresist layer, the photoresist layer substantially shielding the scaffold from light.

[0159] Paragraph A10. The method according to any one of paragraphs A7 to A9, wherein forming the thermosetting resin layer comprises depositing the thermosetting resin onto the hydrogel located in the receiver using a 3D printer.

[0160] Paragraph A11. The method according to paragraph A10, wherein the hydrogel comprises a scaffold supporting organoid formation.

[0161] Paragraph A12. The method according to paragraph A11, wherein the hydrogel comprises a first hydrogel that promotes organoid formation and a second hydrogel that temporarily supports the thermosetting resin layer, and wherein the second hydrogel is configured to substantially melt or dissolve when the chamber is used for organoid culture.

[0162] Paragraph A13. The method according to any one of paragraphs A1 to A3 and A5, wherein the receiver comprises a scaffold to facilitate organoid formation, and wherein sealing comprises forming a thermoplastic material layer on the hydrogel comprising the scaffold and hardening the thermoplastic material layer by cooling to hermetically seal the opening side of the receiver.

[0163] Paragraph A14. The method according to any one of paragraphs A1 to A13, wherein the receiver is defined by a container body, and wherein sealing includes attaching a sealing member to the bottom end of the container body.

[0164] Paragraph A15. The method according to any one of paragraphs A1 to A14, further comprising introducing a fluid and / or at least one substance from the overlying reservoir into the chamber (for contact with the organoid), wherein, optionally, the chamber and the overlying reservoir are integrally formed with each other.

[0165] Paragraph A16. The method according to paragraph A15, wherein the introduction includes transferring fluid and / or at least one substance into the chamber via a channel extending from the overlying reservoir to the chamber, and wherein, optionally, the channel is integrally formed with the overlying reservoir and the chamber.

[0166] Paragraph A17. The method according to paragraph A15 or A16, wherein the introduction includes feeding the organoid by passing nutrients through the top wall of the chamber.

[0167] Paragraph A18. The method according to any one of paragraphs A15 to A17, wherein the introduction includes passing a marker through the top wall of the chamber to mark at least a portion of the organoid.

[0168] Paragraph A19. The method according to any one of paragraphs A15 to A18, wherein the following is introduced:

[0169] (i) passing one or more effectors through the top wall of a chamber, wherein the one or more effectors are selected from the group consisting of: differentiation factors, growth factors, anticancer compounds, expression vectors, viruses, oligonucleotides, messenger RNA, and small interfering RNA; and / or

[0170] (ii) Allowing molecules to pass through the top wall of a chamber, wherein the molecules are configured for genome editing of organoids (e.g., deletion, insertion, or substitution of one or more nucleotides of a target sequence within the organoid cell), such as genome editing using the CRISPR-Cas system, the TALEN system, etc.; and / or

[0171] (iii) To allow molecules to pass through the top wall of a chamber to mark one or more genomic loci of organoids in vivo and / or to alter the expression of one or more genes by binding to those genes, wherein the molecules are based on the CRISPR-Cas system, the TALEN system, etc.

[0172] The molecules described in (ii) or (iii) above may include guide RNA, effector molecules for CRISPR-Cas expression, viruses containing expression vectors of guide RNA and / or CRISPR-Cas system proteins, or any combination of CRISPR-Cas proteins.

[0173] Paragraph A20. The method according to paragraphs A15 or A19, wherein the introduction includes passing one or more fixatives and / or cleaning agents through the top wall of the chamber.

[0174] Paragraph A21. The method according to any one of paragraphs A15 to A20, wherein the introduction includes using gravity to drive fluid from the reservoir into the chamber.

[0175] Paragraph A22. The method according to any one of paragraphs A15 to A21, wherein the introduction includes driving fluid from an inlet to an outlet of a chamber, and wherein the inlet and outlet each include a respective channel extending through the top wall of the chamber.

[0176] Paragraph A23. The method according to any one of paragraphs A15 to A22, wherein the chamber is in independent fluid communication with the first overlying reservoir and the second overlying reservoir, and wherein the introduction includes introducing fluid and / or at least one substance from each of the first overlying reservoir and the second overlying reservoir into the chamber.

[0177] Paragraph A24. The method according to paragraph A23, wherein the organoid defines an internal space located inside it and an external space located outside the organoid and within the cavity, and wherein fluid held by a first overlying reservoir is supplied to the internal space and fluid held by a second overlying reservoir is supplied to the external space.

[0178] Paragraph A25. The method according to any one of paragraphs A15 to A24, wherein the chamber is in independent fluid communication with the first reservoir and the second reservoir, the method further comprising using a pump to transfer fluid from the second reservoir to the first reservoir to facilitate a gravity-driven flow from the first reservoir through the chamber to the second reservoir.

[0179] Paragraph A26. According to the method described in paragraph A25, the pump transfers fluid from the second reservoir to the first reservoir at a rate substantially matching the rate of the gravity-driven flow.

[0180] Paragraph A27. The method according to paragraph A26, wherein the chamber is in independent fluid communication with a first reservoir and a second reservoir defined by a container body, the method further comprising repeatedly tilting the container body to alternately generate gravity-driven flows from the first reservoir to the second reservoir and from the second reservoir to the first reservoir.

[0181] Paragraph A28. The method according to any one of paragraphs A15 to A27, wherein the first reservoir and the second reservoir are in fluid communication with the chamber via respective first and second channels, wherein the first reservoir and the second reservoir are in direct fluid communication with each other via a third channel above and spaced apart from the top wall of the chamber.

[0182] Paragraph A29. The method according to any one of paragraphs A15 to A28, wherein the first reservoir and the second reservoir are located above the chamber and are in independent fluid communication with the chamber, the method further comprising disposing a flexible membrane above the first reservoir and applying pressure to the top side of the flexible membrane to drive fluid from the first reservoir through the chamber to the second reservoir.

[0183] According to paragraph A30. According to the method described in paragraph A29, wherein setting the flexible membrane includes setting the flexible membrane above each of the first reservoir and the second reservoir, and wherein applying pressure includes alternately applying pressure to the top side of the flexible membrane above the first reservoir and to the top side of the flexible membrane above the second reservoir to alternately drive the fluid between the first reservoir and the second reservoir in opposite directions.

[0184] Paragraph A31. The method according to any one of paragraphs A1 to A30 further includes placing a scaffold within the receiver before sealing the open side of the receiver, the scaffold being configured to promote organoid formation.

[0185] Paragraph A32. The method according to paragraph A31, wherein setting the support includes forming the support within the receiver.

[0186] Paragraph A33. According to the method described in paragraph A31, setting the support includes placing a prefabricated support in the receiver.

[0187] Paragraph A34. The method according to any one of paragraphs A31 to A33 further includes introducing biological cells into the receiver before sealing the open side of the receiver.

[0188] Paragraph A35. According to the method described in paragraph A34, the biological cells include stem cells.

[0189] Paragraph A36. The method described in paragraphs A34 or A35, wherein biological cells are introduced into the scaffold during scaffold formation.

[0190] Paragraph A37. The method according to any one of paragraphs A32 and A34 to A36, wherein the scaffold is formed by 3D printing.

[0191] Paragraph A38. The method described in paragraph A37, wherein forming the scaffold includes 3D printing at least two different hydrogels in the receiver.

[0192] Paragraph A39. The method according to paragraph A38, wherein at least one hydrogel contains cells during 3D printing to achieve a 3D scaffold of arbitrary shape that promotes the formation of a specific organoid.

[0193] Paragraph A40. The method according to any one of paragraphs A31 to A39, wherein the receiver has a wall opposite to the opening side, and wherein the support is attached to the wall.

[0194] Paragraph A41. The method according to any one of paragraphs A31 to A40 further includes introducing the biological cells for forming the organoid into the chamber after sealing the opening side of the receiver to form the chamber.

[0195] Paragraph A42. The method according to any one of paragraphs A31 to A41, wherein the chamber has a top wall, and wherein the organoid is supported by the top wall during formation.

[0196] Paragraph A43. The method according to any one of paragraphs A31 to A42, wherein the chamber is in fluid communication with a plurality of overlying reservoirs via channels, the method further comprising forming extensions of one or more channels by 3D printing, and optionally embedding the extensions in a hydrogel.

[0197] Paragraph A44. The method according to paragraph A43 further includes extending and branching each extension by 3D printing to form one or more laterally permeable tubes for supplying material to cells inside the hydrogel.

[0198] Paragraph A45. The method according to paragraph A44 further includes supplying one or more effectors via one or more transversely permeable tubes to establish one or more concentration gradients of the effector within a chamber for more controlled stem cell differentiation and organoid formation.

[0199] Paragraph A46. The method according to any one of paragraphs A43 to A45 further includes incorporating polymer or metal microtubes into the hydrogel to facilitate fluid flow.

[0200] Paragraph A47. The method according to any one of paragraphs A1 to A46, further comprising collecting organoid-related data while the organoid remains in the chamber.

[0201] Paragraph A48. The method described in paragraph A47, wherein collecting data includes capturing images of at least a portion of the organoid.

[0202] Paragraph A49. The method according to paragraph A48, wherein capturing includes capturing an image by light sheet microscopy.

[0203] Paragraph A50. The method according to paragraph A48 or A49, wherein capturing includes detecting photoluminescence from organoids.

[0204] Paragraph A51. The method according to any one of paragraphs A48 to A50, wherein capturing includes capturing an image stack representing at least a portion of a 3D structure of an organoid.

[0205] Paragraph A52. The method according to paragraph A51, wherein capturing includes irradiating at least a portion of the organoid via a transverse window of the chamber, and detecting optical radiation propagating out of the chamber via a bottom window of the chamber, and vice versa.

[0206] Paragraph A53. The method according to any one of paragraphs A47 to A52, wherein collecting data includes analyzing fluid from the chamber or overlying compartment for the analyte.

[0207] According to paragraph A54. According to any one of paragraphs A47 to A53, data collection is performed using a sensor at least partially located within the chamber.

[0208] Paragraph A55. The method according to any one of paragraphs A47 to A54, wherein data collection includes collecting data on cells and / or fluids removed from the chamber while the bottom of the chamber remains sealed.

[0209] Paragraph A56. The method according to paragraph A55, wherein collecting data includes forming an opening in the top wall of the chamber and removing cells and / or fluid from the chamber via the opening.

[0210] Paragraph A57. The method according to any one of paragraphs A1 to A56, the method further comprising forming an opening in the wall of the chamber; and inserting an end of an instrument into the chamber through the opening.

[0211] Paragraph A58. The method according to paragraph A57, wherein a plurality of overlying compartments and chambers share a common wall, and wherein an opening is formed in the common wall at the bottom end of one of the overlying compartments.

[0212] Paragraph A59. The method according to paragraph A58, wherein the plurality of overlying compartments include a plurality of reservoirs and one or more inlet pipes, and wherein an opening is formed at the bottom end of one of the inlet pipes.

[0213] Paragraph A60. The method according to paragraph A58 or A59, wherein an opening is formed using an instrument, and wherein forming the opening includes breaking a common wall using the end of the instrument.

[0214] Paragraph A61. The method according to any one of paragraphs A57 to A60, wherein the end of the instrument is a sharp end.

[0215] Paragraph A62. The method according to paragraphs A60 or A61, wherein forming an opening includes forming a fluid tight seal between the device and the common wall at the opening.

[0216] Paragraph A63. The method according to any one of paragraphs A60 to A62, wherein the common wall is defined as having the characteristic that the common wall is constructed to be torn by mechanical pressure applied to the common wall via an instrument.

[0217] Paragraph A64. The method according to any one of paragraphs A57 to A63, wherein the instrument is selected from the group consisting of: needles, light guides operatively connected to a light source, endoscopes, electrodes, ATR probes, pneumatic / hydraulic sources optionally connected to a sac, and magnets.

[0218] Paragraph A65. The method according to any one of paragraphs A57 to A64, wherein the apparatus includes a sensor located at its end.

[0219] Paragraph A66. The method described in paragraph A65 further includes using sensors to sense parameters of the chamber and / or organoid.

[0220] Paragraph A67. The method according to any one of paragraphs A57 to A66, wherein the device includes electrodes configured to electrically stimulate organoids.

[0221] Paragraph A68. The method described in paragraph A67 further includes using electrodes to electrically stimulate the organoid.

[0222] Paragraph A69. The method according to any one of paragraphs A57 to A68, wherein the apparatus includes a light guide, the light guide being optically coupled to a light source and having an aperture at its end that is introduced into the chamber.

[0223] Paragraph A70. The method according to any one of paragraphs A57 to A69, wherein the instrument includes a magnet located at an end in the introduction chamber.

[0224] Paragraph A71. The method according to any one of paragraphs A57 to A70, wherein the instrument includes an attenuated total reflection (ATR) probe.

[0225] Paragraph A72. The method according to any one of paragraphs A57 to A71, wherein the instrument includes an endoscope.

[0226] Paragraph A73. The method according to any one of paragraphs A57 to A72, wherein the apparatus is connected to a pneumatic or hydraulic source.

[0227] Paragraph A74. The method according to any one of paragraphs A1 to A73 further includes introducing the test compound into the chamber.

[0228] Paragraph A75. The method according to any one of paragraphs A1 to A74, wherein the method includes forming a plurality of organoids in respective plurality of chambers.

[0229] Paragraph A76. The method described in paragraph A75 also includes applying different treatments to each organoid.

[0230] Paragraph A77. The method according to paragraph A76, wherein applying different treatments includes introducing different test compounds into each of the multiple chambers.

[0231] Paragraph A78. The method described in paragraph A77, wherein each different test compound is a potential anticancer drug.

[0232] Paragraph A79. The method according to any one of paragraphs A76 to A78 further includes introducing one or more cells (optionally cancer cells) into each chamber.

[0233] Paragraph A80. The method according to paragraph A79, wherein, optionally, one or more cells are introduced into the organoid in the chamber via a needle, wherein optionally, the needle punctures the top wall of the chamber.

[0234] Paragraph A81. The method according to any one of paragraphs A76 to A80 further includes collecting data for each of a plurality of organoids to test the effects of different treatments on the organoids.

[0235] Paragraph A82. The method according to paragraph A81, wherein data collection includes in situ imaging of each organoid in its respective chamber.

[0236] Paragraph A83. The method described in paragraphs A81 or A82, wherein collecting data includes analyzing the corresponding fluids associated with each organoid for the analyte.

[0237] Paragraph A84. The method according to any one of paragraphs A81 to A83, wherein collecting data includes optionally removing the organoid from the chamber of each organoid after removing the sealing member.

[0238] Paragraph A85. The method according to paragraph A84, wherein collecting data includes imaging each organoid after removing the organoid from the chamber of each organoid.

[0239] Paragraph A86. The method according to paragraph A85, wherein collecting data includes physically slicing each organoid after removing the organoid from the chamber of each organoid; and optionally, imaging multiple slices generated by the physical slicing.

[0240] Paragraph B1. A method for culturing and / or analyzing organoids, the method comprising: (a) forming an organoid within a chamber, wherein an access tube is located above the chamber, and wherein the bottom end of the access tube is closed by a top wall region of the chamber; (b) supplying nutrients to the chamber to feed the organoid; (c) forming an opening through the top wall region; and (d) inserting an end of an instrument from the access tube into the chamber.

[0241] Paragraph C1. A method for culturing and / or analyzing organoids, the method comprising: (a) sealing the open side of a receiver to form a chamber; (b) forming an organoid within the chamber; (c) supplying a substance / fluid into the chamber; and (d) capturing an image of at least a portion of the organoid while the organoid remains enclosed by the walls of the chamber.

[0242] Paragraph D1. An apparatus for culturing and / or analyzing organized multicellular structures (e.g., organoids), the apparatus comprising: (a) a body defining a receiver and at least two reservoirs disposed on the receiver and in independent fluid communication with the receiver via respective channels; and (b) a sealing member coupled to or combustable to the body at an open side of the receiver to form a chamber for a multi-unit structure.

[0243] Paragraph D2. The apparatus according to paragraph D1 further includes a scaffold for attaching biological cells to the wall of the receiver.

[0244] Paragraph D3. The device according to paragraph D2, wherein the scaffold is included in the hydrogel.

[0245] Paragraph D4. The apparatus according to paragraph D3, wherein the hydrogel contains biological cells.

[0246] Paragraph D5. The apparatus according to paragraph D4, wherein the biological cells include stem cells.

[0247] Paragraph D6. The apparatus according to any one of paragraphs D3 to D5, wherein the hydrogel contains a deposit of a drug.

[0248] Paragraph D7. The apparatus according to paragraph D6, wherein the deposit can be opened by photoinduced release to release a drug from the deposit.

[0249] Paragraph D8. The apparatus according to any one of paragraphs D1 to D7, wherein the receiver and at least two reservoirs are integrally formed with each other.

[0250] Paragraph D9. The apparatus according to any one of paragraphs D1 to D8, wherein the main body is injection molded as a single piece.

[0251] Paragraph D10. The apparatus according to any one of paragraphs D1 to D9, wherein each of the at least two reservoirs shares a wall with the receiver.

[0252] Paragraph D11. The apparatus according to any one of paragraphs D1 to D10, wherein each channel extends through a wall located between the respective reservoir and the receiver, and the wall is optionally shared between the respective reservoir and the receiver.

[0253] Paragraph D12. The device according to any one of paragraphs D1 to D11 further includes a removable cover configured to cover the top of the opening of each of at least two reservoirs.

[0254] Paragraph D13. The apparatus according to any one of paragraphs D1 to D12, wherein the apparatus is under sterilization conditions.

[0255] Paragraph D14. The device according to any one of paragraphs D1 to D13, wherein the sealing member is attached to the body.

[0256] Paragraph D15. The apparatus according to paragraph D14, wherein an organoid is contained in a chamber.

[0257] Paragraph D16. The apparatus according to any one of paragraphs D1 to D15, wherein the body defines at least four reservoirs, each reservoir being configured to be in fluid communication with the receiver.

[0258] Paragraph D17. The apparatus according to any one of paragraphs D1 to D16, wherein the sealing member is configured to provide a bottom window for imaging at least a portion of the organoid housed in the chamber.

[0259] Paragraph D18. The apparatus according to any of paragraphs D1 to D17, wherein the body is provided with a pair of transverse windows to allow illumination of at least a portion of the organoid contained in the chamber via either transverse window.

[0260] Paragraph D19. The apparatus according to any one of paragraphs D1 to D18, wherein the body defines an inlet tube having an open top end and a closed bottom end, and wherein a common wall separates the chamber from the bottom end of the tube.

[0261] Paragraph D20. The apparatus according to any one of paragraphs D1 to D19 further includes a linear array of substantially identical units connected to each other, wherein one of the units includes a receiver and at least two reservoirs.

[0262] As used in this disclosure, the term "exemplary" means "illustrative" or "as an example." Similarly, the term "illustrative" means "to illustrate by way of example." This term does not imply desirability or superiority.

[0263] The above disclosure may include several different inventions with independent utility. Although each of these inventions is disclosed in its preferred form, the specific embodiments disclosed and shown herein should not be considered limiting, as many variations are possible. The subject matter of this invention includes all novel and non-obvious combinations and sub-combinations of the various elements, features, functions, and / or characteristics disclosed herein. The appended claims specifically point to certain combinations and sub-combinations that are considered novel and non-obvious. Inventions implemented in other combinations and sub-combinations of features, functions, elements, and / or characteristics may be claimed in applications claiming priority to this application or related applications. Whether for different or the same invention, and whether broader, narrower, equal to, or different from the scope of the original claims, such claims are considered to be included within the subject matter of the invention disclosed herein. Furthermore, unless expressly stated otherwise, sequence indicators such as first, second, or third for identified elements are merely for distinguishing elements and do not indicate a specific location or order of these elements.

Claims

1. A method for culturing organoids, the method comprising: Set the receiver; where: The receiver includes a top wall and a pair of transverse walls, the top wall having a first end and a second end, and each of the pair of transverse walls having a first end and a second end; The first end of the first transverse wall of the pair of transverse walls extends from the first end of the top wall, and the first end of the second transverse wall of the pair of transverse walls extends from the second end of the top wall; and The second end of the first transverse wall in the pair of transverse walls does not contact the second end of the second transverse wall in the pair of transverse walls; The receiver and the reservoir integrally formed with the receiver are positioned in an inverted position, in which the receiver is disposed above the reservoir; The bracket is mounted on the top wall of the receiver; A sealing member is joined to each of the second ends of the first transverse wall of the pair of transverse walls of the receiver and the second end of the second transverse wall of the pair of transverse walls of the receiver to form a chamber; The chamber and the reservoir are inverted such that the chamber is positioned below the reservoir; and The scaffold is used to form organoids in the chamber.

2. The method according to claim 1, wherein, The bonding includes bonding a prefabricated sealing member to the receiver.

3. The method according to claim 1, wherein, The bonding process includes forming the sealing member by at least partially curing a sealing material in the receiver.

4. The method of claim 1, further comprising introducing fluid and / or at least one substance from the reservoir integrally formed with the receiver into the chamber.

5. The method according to claim 1, wherein, Setting up the bracket involves 3D printing the bracket in the receiver.

6. The method of claim 1, further comprising capturing an image of at least a portion of the organoid when the organoid is located in the chamber.

7. The method according to claim 6, wherein, The capture includes capturing images using light sheet microscopy.

8. A method for culturing organoids, the method comprising: Set the receiver; where: The receiver includes a top wall and a pair of transverse walls, the top wall having a first end and a second end, and the pair of transverse walls each having a first end and a second end; The first end of the first transverse wall of the pair of transverse walls extends from the first end of the top wall, and the first end of the second transverse wall of the pair of transverse walls extends from the second end of the top wall; and The second end of the first transverse wall in the pair of transverse walls does not contact the second end of the second transverse wall in the pair of transverse walls; The receiver and the reservoir integrally formed with the receiver are positioned in an inverted position, in which the receiver is disposed above the reservoir; The bracket is mounted on the top wall of the receiver; A sealing member is attached to each of the second ends of the first transverse wall of the pair of transverse walls of the receiver and the second transverse wall of the pair of transverse walls of the receiver to form a chamber; The chamber and the reservoir are inverted such that the chamber is positioned below the reservoir; and When the sealing member is attached to each of the second ends of the first transverse wall of the pair of transverse walls of the receiver and the second end of the second transverse wall of the pair of transverse walls of the receiver, the scaffold is used to form an organoid in the chamber.

9. The method according to claim 8, wherein, Setting up the bracket involves 3D printing the bracket in the receiver.

10. The method according to claim 9, wherein, The 3D printing includes incorporating stem cells into the scaffold.

11. The method according to claim 8, wherein, The attachment of the sealing member includes a second end of the second end of the first transverse wall of the pair of transverse walls of the receiver, and a second end of the second transverse wall of the pair of transverse walls.

12. The method according to claim 8, further comprising: An opening is formed in the top wall of the chamber; as well as Insert the end of the instrument into the chamber through the opening.

13. The method according to claim 12, wherein, The instrument is selected from the group consisting of: needles, light guides operatively connected to a light source, endoscopes, electrodes, ATR probes, pneumatic / hydraulic sources connected to a sac, and magnets.

14. A method for culturing organoids, the method comprising: Set the receiver; where: The receiver includes a top wall and a pair of transverse walls, the top wall having a first end and a second end, and the pair of transverse walls each having a first end and a second end; The first end of the first transverse wall of the pair of transverse walls extends from the first end of the top wall, and the first end of the second transverse wall of the pair of transverse walls extends from the second end of the top wall; and The second end of the first transverse wall in the pair of transverse walls does not contact the second end of the second transverse wall in the pair of transverse walls; The receiver and the reservoir are positioned in an inverted position, in which the receiver is positioned above the reservoir; The bracket is mounted on the top wall of the receiver; A sealing member is attached to each of the second ends of the first transverse wall of the pair of transverse walls of the receiver and the second transverse wall of the pair of transverse walls of the receiver to form a chamber; The chamber and the reservoir are inverted such that the chamber is positioned below the reservoir; Organoids are formed in the chamber using the scaffold; and A fluid and / or at least one substance is introduced from the reservoir integrally formed with the receptor through the top wall into the chamber to contact the organoid.

15. The method according to claim 14, wherein, The introduction includes allowing the fluid and / or at least one substance to enter the chamber via a channel that extends through a wall located between the reservoir and the chamber.

16. The method of claim 14, wherein, The introduction includes feeding the organoid by passing nutrients through the top wall of the chamber.

17. The method of claim 14, wherein, The introduction includes passing one or more fixatives and / or desiccants through the top wall of the chamber.

18. The method according to claim 14, wherein, The introduction includes using gravity to drive the fluid from the reservoir into the chamber.

19. The method of claim 14, wherein, The introduction includes driving the fluid from the inlet to the outlet of the chamber, and each of the inlet and the outlet includes a channel extending through the top wall of the chamber.

20. The method of claim 14, further comprising: Multiple organoids are formed in the corresponding multiple chambers; as well as Different treatments are applied to each of the plurality of organoids.

21. The method according to claim 20, wherein, Applying the different treatments involves introducing different test compounds into each of the plurality of chambers.

22. The method according to claim 20, wherein, Applying these different treatments involves altering the genetic material in the corresponding chambers of each organoid through genome editing.

23. The method according to claim 20, wherein, Applying the different treatments includes in vivo labeling of at least one genomic locus of each organoid in the respective compartment of each organoid using one or more DNA binding agents or altering the gene expression profile of each organoid.

24. The method of claim 20, further comprising collecting data for each of the plurality of organoids to test the effects of the different treatments on the plurality of organoids.

25. The method according to claim 24, wherein, The data collection includes in-situ imaging of each organoid in the corresponding chamber of each organoid.

26. The method according to claim 24, wherein, Collecting the data includes analyzing the corresponding fluids associated with each organoid for the analyte.

27. The method according to claim 24, wherein, Collecting the data includes removing each organoid from the chamber of each organoid, and collecting the data from the organoid after removing each organoid from the chamber of each organoid.

28. The method of claim 27, wherein, Collecting the data includes physically slicing each organoid after removing it from the chamber of each organoid.

29. The method according to claim 14, wherein, The first reservoir and the second reservoir are positioned on the chamber and are in independent fluid communication with the chamber. The method further includes placing a flexible membrane above the first reservoir and applying pressure to the top side of the flexible membrane to drive the fluid from the first reservoir through the chamber to the second reservoir.

Images