Systems and methods for organoid culture

Abstract

The present disclosure provides systems, including methods and devices, for culturing, monitoring and / or analyzing organoids. In an exemplary method of organoid culture, the method may include disposing a stent in a receptacle having an open side. A sealing member may be bonded to the open side of the receptacle to form a chamber. A stent may be used to form an organoid in the chamber. A fluid and / or at least one substance may be introduced from the overlying reservoir into the chamber to contact the organoid.

Description

[0001] This application is a divisional application of the patent application with application number 2020800422416, application date May 7, 2020, and invention name “Systems and methods for organoid culture”.

[0002] Related Applications

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

[0004] Organoids ("mini-organs") are three-dimensional clumps of different types of cells generated in vitro and have some similarities to organs, such as showing actual histology of organ-specific organization. Clumps of cells can be produced 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. With this approach, organoids resembling tissues from the brain, heart, intestine, kidney, liver and stomach, among others, have been produced to date. These promising results suggest that organoid cultures have the potential to provide new insights into organ development and function, and to recreate disease models that allow for in vitro drug screening. Organoids could revolutionize how drugs are discovered and personalized.

[0005] There are many problems that limit the ability of researchers to fully develop organoids. First, along with the increase of organoid size, organoids may need to be fed from both inside and outside, which presents a challenge. Secondly, each different type of organoid will require optimized three-dimensional matrix scaffold structure, culture medium exchange (feeding) suitable for the structure, and may even need to withstand the ability of mechanical resistance or controlled force, to allow the appropriate growth and development of functional organoids. The third, there is no container for allowing screening and optimizing large organoids to be exposed to different types of reagents. The fourth, there is no container optimized for in-situ monitoring of large organoids by imaging methods. On the contrary, organoids are usually imaged after they are fixed and physically sliced. Therefore, it is necessary to improve the system and method for organoid culture, such as improving organoid culture by providing the ability of effective growth, monitoring and analyzing large organoids. Summary of the invention

[0006] The present disclosure provides systems for culturing, monitoring and / or analyzing organoids or other organized multicellular structures, including methods and devices. In an exemplary method of organoid culture, the method may include arranging a support in a receptacle having an open side. A sealing member may be coupled to the open side of the receptacle to form a chamber. A support may be used to form an organoid in the chamber. A fluid and / or at least one substance may be introduced into the chamber from an overlying reservoir to contact the organoid. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0009] Figure 3 yes Figure 2 A side view of the body of the container, which is as shown in Figures 4 to 10 The methods for culturing, monitoring and / or analyzing organoids in a container are shown in the drawings and are shown in FIG. Figure 2 Inverted.

[0010] Figure 4 and Figure 5 yes Figure 3 Side views of a vessel body, drawn during and after forming a scaffold in a receptacle of the vessel by 3D printing or pipetting.

[0011] Figure 6 yes Figure 5 A side view of a container body after a preformed sealing member has been bonded to the container body to seal the open side of the receptacle, thereby creating a chamber containing the stent.

[0012] Figure 7 yes Figure 6 A side view of a container body and sealing member 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 a lid of the container has covered an open top of the reservoir.

[0013] Figure 8 The images were drawn after stem cells in the scaffold proliferated and differentiated to generate organoids. Figure 7 Side view of the container.

[0014] Fig. 9 The images were drawn after remodeling / replacing the scaffold by allowing organoids to develop Figure 8 Side view of the container.

[0015] Fig.10 This is what happens when organoids are imaged using light-sheet microscopy. Fig. 9 Side view of the container.

[0016] Figures 11 to 13yes Figure 5 A side view of a container body and a support showing an alternative method of sealing the open side of the receptacle of the container body to form a chamber by forming a sealing member in situ in the receptacle.

[0017] Fig.14 yes Fig.13 A side view of a container body, sealing member, and scaffold, drawn in the presence of 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 scaffold and into an overlying reservoir in fluid communication with the chamber, and a lid of the container has covered the open top of the reservoir.

[0018] Fig.15 yes Fig.12 A side view of the container body and the bracket, showing the relative Fig.13 An improved method of sealing an open side of a receptacle of a container body to form a chamber by forming a sealing member in situ in the receptacle.

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

[0020] Fig.17 yes Fig.16 Schematic side view of a container strip drawn during organoid culture and showing an exemplary pump configuration for transferring liquid culture medium between reservoirs within each container of the container strip.

[0021] Fig.17A and Fig. 17B Shows Fig.16 Schematic side view of a container strip drawn during organoid culture and showing how tilting the container strip in opposite rotational directions can drive flow in respective opposite directions within each chamber.

[0022] Fig.18 is a schematic side view of another embodiment of an exemplary container strip having an array of containers drawn during organoid culture and showing an exemplary pump configuration for transferring liquid culture medium between reservoirs of different containers of the container strip.

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

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

[0025] Fig.21 yes Fig.19 A view of a container body of the invention, drawn with the container body inverted and the preformed sealing member exploded from the receptacle of the container body.

[0026] Fig. 22 yes Fig.19 The container body is roughly along Fig. 20 A bottom view taken along line 22-22.

[0027] Fig.23 yes Fig.19 The container body is roughly along Fig. 20 A top view taken along line 23-23.

[0028] Fig.24 yes Fig.19 The container body is roughly along Fig.23 A cross-sectional view taken along line 24-24.

[0029] Fig.25 yes Fig.19 The container body is roughly along Fig.23 Another cross-sectional view taken along line 25-25.

[0030] Fig.26 Another embodiment of the container body is as follows Fig.25 The partial cross-sectional view taken in this way shows only the container body which is different from Fig.25 The lower part.

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

[0032] Fig.28 Another embodiment of the container body is as follows Fig.25 A cross-sectional view taken in such a manner, wherein the container body includes an electromagnet operably connected to a receptacle of the container body.

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

[0034] Fig.30 yes Fig.29 Another partial view of the container body, sealing member, support and cells, which is drawn when the electromagnet is turned on so that the ferromagnetically labeled cells are pulled into the support by magnetic force.

[0035] Fig.31is a top view of an exemplary rack of containers arranged in a linear pattern as a strip held during scaffold formation and organoid formation and culture, wherein each container includes Fig.19 The container body corresponding to the embodiment.

[0036] Fig.32 yes Fig.31 The assembly of the shelf is Fig.19 A top view of a set of container bodies corresponding to an embodiment to form a strip.

[0037] Fig.33 yes Fig.32 Side view of the strip.

[0038] Fig.34 is to maintain Fig.19 A top view of another exemplary rack of an embodiment corresponding to a two-dimensional array of container bodies to form strips.

[0039] Fig.35 is used for Figure 1 and Figure 2 Another embodiment of the container body of the container is substantially as follows Fig.25 (for related embodiments) in which the open bottom end of the container body is sealed by a sealing member to form a chamber, wherein the container body defines an entry tube, which is closed at the bottom end of the entry tube by a rupturable barrier, and wherein a magnet is inserted into the entry tube to place the working end of the magnet near (and above) the rupturable barrier.

[0040] Fig.36 yes Fig.35 A cross-sectional view of a container body and sealing member taken with an organoid present in the chamber and the magnet replaced by a needle having a pointed end that pierces the rupturable barrier and enters the organoid.

[0041] Fig.37 yes Fig.35 A cross-sectional view of a container body and sealing member taken with an organoid present in the chamber and the magnet replaced by a sensor / electrode having a sensing / stimulation end region that pierces the rupturable barrier and enters the organoid.

[0042] Figures 38 to 40 yes Fig.35 A partial cross-sectional view of a container body of FIG. 1 , taken around the rupturable barrier before, during, and after the tip of the rupturing device passes through the barrier and enters the chamber from the access tube.

[0043] Fig.41 yes Fig.35 The container body and the sealing member are substantially as follows Fig.24The cross-sectional view is taken as described above (for the relevant embodiment) so that three access tubes are visible, wherein rupture instruments are arranged in the three access tubes, and wherein only one rupture instrument extends into the chamber below the access tube.

[0044] Figure 42 to Figure 44 yes Fig.35 Partial cross-sectional views of a variant embodiment of a container body in which the rupturable barrier is configured to tear at a frangible web, wherein the views are taken around the rupturable barrier before, during, and after the tip of the rupturing device passes through the rupturable barrier from the access tube.

[0045] Fig.45 yes Fig.35 A cross-sectional view of a container body and sealing member taken with an organoid present in the chamber and the magnet replaced by an attenuated total reflection (ATR) fiber optic probe having a tip that passes through the rupturable barrier and enters the organoid.

[0046] Fig.46 yes Fig.35 A cross-sectional view of the container body and sealing member taken with an organoid present in the chamber and the magnet replaced by an imaging fiber optic probe with a front lens passing through the rupturable barrier and into the organoid.

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

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

[0049] Fig.49 yes Fig.35 The container body and the sealing member are as follows Fig.24 (for related embodiments) wherein the organoid is present in the chamber and the magnet is replaced by a pair of pneumatic devices having an inflatable balloon located outside the organoid to generate external mechanical strain.

[0050] Fig.50 and Fig.51 yes Fig.35 The container body and the sealing member are respectively substantially as follows Fig.25 and Fig.24A view taken as in the example above (for a similar embodiment) in which a pair of magnets extend into the chamber via corresponding access tubes and a prefabricated support structure or biochip is mounted onto the magnets via magnetic attraction.

[0051] Fig.52 and Fig.53 yes Fig.35 A cross-sectional view of a container body and a sealing member, taken in the following situation: a cover is installed on the top of the container body and seals the top of the container body, and a flexible member is provided, which is deformed by external pressure alternately applied to a pair of reservoirs to drive liquid to pass through the chamber alternately in opposite directions. DETAILED DESCRIPTION

[0052] The present disclosure provides a system, including methods and apparatus, for 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. In an exemplary method of organoid culture, the method may include placing a scaffold in a receptacle having an open side. A sealing member may be coupled to the open side of the receptacle to form a chamber. The scaffold may be used to form an organoid in the chamber. A fluid and / or at least one substance may be introduced into the chamber from an overlying reservoir to contact the organoid.

[0053] The present 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, etc. The container can be consumable (i.e., disposable after a single use) and / or can have a standard shape. The container can include a container body that defines a receptacle with an open side. A sealing member can be attached (e.g., combined) to the container body to form a chamber by the receptacle. The sealing member can be preformed or formed in a receptacle, etc.

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

[0055] Suitable cells that can be introduced into the matrix scaffold, either while forming the matrix scaffold or after the matrix scaffold has been formed, can include undifferentiated stem cells, stem cells that have differentiated and will continue to differentiate, cell aggregates, small organoids, and the like.

[0056] The 3D printer can also be used to apply an adhesive and / or sealing fluid 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 technologies that can be suitable for dispensing matrix components include droplet-based bioprinting using bio-inks, or laser-based bioprinting (e.g., laser-based direct writing of printing cells, enzymes, etc. by laser-induced forward transfer (LIFT) using a pulsed laser).

[0057] Scaffold and / or other matrix can be provided by one or more hydrogels.Every kind of hydrogel can comprise one or more thermoplastic structural components, such as matrigel, alginate, nanofibrillary cellulose (nanofibrillarcellulose), collagen, fibrin and / or polyethylene glycol etc., and these one or more thermoplastic structural components form matrix in a temperature-dependent manner in a coordinated manner.

[0058] In some embodiments, two or more different hydrogels / matrices may be disposed in a receptacle. The hydrogels / matrices may differ by any suitable parameter, such as melting temperature, resistance to enzymatic degradation, solubility, cell attraction and / or cell repulsion properties, and the like.

[0059] Each hydrogel / matrix can 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, keratan sulfate, etc.), proteoglycans (e.g., GAGs connected to core proteins (such as by their serines) to form the following material: aggrecan, aggrecan, short fibrous proteoglycan, type XVIII collagen, dandruff protein, neurocan, basement membrane proteoglycan, leucine-rich small proteoglycans, versican, etc.), fibrin (e.g., collagen, elastin, fibronectin, laminin, etc.) and / or the like. Protease recognition sites (e.g., for scaffold metalloproteinases (MMPs)) can be incorporated in hydrogels / matrix to allow degradation / remodeling by cells. The frequency of these sites and the order of each site can be selected to allow the appropriate amount of degradation / remodeling.

[0060] One or more growth factors can be included in the matrix when the matrix is ​​formed, or can be introduced into the liquid culture medium after the liquid culture medium is formed. Suitable exemplary growth factors include angiogenin, bone morphogenetic protein (BMP), ciliary neurotrophic factor, colony stimulating factor, ephrins, 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, neuregulin, neurotrophin, platelet-derived growth factor, transforming growth factor, tumor necrosis factor (α), vessel endothelial growth factor, etc.

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

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

[0063] The printed 3D structures are able to provide a temporary scaffold for the appropriate type of cells while they develop into organoids. The cells can self-organize and produce their own extracellular matrix, which can replace some or all of the scaffold. The same is true for internal feeding: the container can provide a universal interface that can optionally be modified through 3D printing, and the cells can organize to optimally use that modified interface.

[0064] While the container is inverted, the scaffold (with or without cells) can be placed in the receptacle of the container body, and the chamber can be formed by the receptacle. Once these processes are completed, the container can be turned right side up (to its organoid culture orientation), and at least one reservoir placed above the chamber can be filled with feed liquid. If there are no cells in the scaffold yet, suitable cells can be placed in the feed liquid, and these suitable cells can be introduced into the scaffold from the overlying reservoir together with the feed liquid (or the cells can be introduced via an entry tube (e.g., see Example 7)).

[0065] Formation of organoids may require an initial culture time before a specific feeding regimen can be initiated. A feeding regimen may include loading a reservoir with appropriate culture medium and removing culture medium from the reservoir according to a predetermined plan and / or based on the developmental stage or condition of the organoid. A feeding regimen may depend on the shape of the scaffold and the type of organoid to be formed.

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

[0067] The container can achieve light sheet 3D imaging. The chamber of the container can have two, three or more optical windows, and light can propagate into and / or out of the chamber via each window. For example, the container can have a bottom window and one or more lateral windows that can all be planar. In some embodiments, the container can have a pair of lateral windows arranged relative to each other.

[0068] A plurality of container bodies (and containers) can be organized as strips. The strips can be formed by pre-connecting the container bodies to each other in a linear or two-dimensional array during the manufacturing process (e.g., by integrally combining or forming the container bodies to each other, such as by injection molding). Alternatively, the strips can be formed during the manufacturing process, or formed by the user by assembling the various container bodies with suitable strip holders. In some embodiments, the strip holder can be configured to hold only one strip or two strips to allow space for an imaging objective. The strips of the container bodies can be loaded into a 3D printer to allow the printer to print a bracket in each container body and / or add a sealing member to each container body.

[0069] The present disclosure can produce large functional organoids by feeding the organoids with different culture media inside and outside. The average diameter or maximum diameter of large organoids can be greater than about 0.1, 0.2, 0.5, 1 or 2 millimeters, etc. The study of large organoids is still challenging, and researchers face two major limitations. First, each type of organoid requires different culture conditions, such as specific hydrogels, matrix as substrate or even mechanical properties (such as shear force caused by culture medium flow). Secondly, the microscopy of large organoids is very challenging. The prior art method is still to thinly slice the organoid material, stain, and use a confocal scanning microscope or even a slide reader to obtain images of fixed samples.

[0070] The present disclosure provides a system and method that helps to overcome one or two obstacles. By using a combination of 3D printing (scaffolds and / or cells) and culture medium exchange by gravity flow, users can generate a unique 3D environment optimized for each type of organoid. Various different types of organoids can be grown. Feeding and waste removal can be solved by fluid communication between the chamber of the container and the overlying reservoir. Optical windows are integrated into each container, at least one optical window is used for the entry of excitation light and another window is used for the departure of emitted light, allowing the living cells of organoids to be monitored by light sheet microscopy. Alternatively or additionally, organoids can be imaged via one or more windows by classical wide-field microscopy. Therefore, the container disclosed herein can make it possible to implement live cell microscopy to developing and / or developed organoids. High capacity and / or high throughput microscopy can be implemented to organoids.

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

[0072] (I) containers for organoid formation, culture, monitoring and / or analysis, (II) methods for organoid formation, culture, monitoring and / or analysis, and / or (III) examples.

[0073] I. Containers 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 an organoid 52 (or other organized multicellular structure); see Figure 1 and Figure 2 The container 50 is schematically shown here, with the front and rear walls of the container not visible to distinguish the closure of the container ( Figure 1 ) or open ( Figure 2 )'s top and bottom sides.

[0075] Figure 1 A container 50 is shown containing an organoid 52 in 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 can be attached to an upper region of the chamber 54, such as a top wall 58 of the chamber (the top wall can be interchangeably referred to as a top plate). The transverse walls 60 and bottom wall 62 of the chamber can provide a barrier to the passage of fluid, thereby making all of the chamber below the top wall 58 fluid-tight.

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

[0077] The two or more reservoirs of the container 50 can hold any suitable substance to be supplied to the chamber 54. Exemplary substances include nutrients, effectors, reagents, etc. Suitable nutrients include any substance that promotes cell health and proliferation in the chamber 54 and thus promotes the growth and development of the organoid 52. Exemplary nutrients may include sugars (such as glucose), amino acids, proteins, nucleotides, vitamins, minerals, fatty acids, etc. Effectors include any molecules (such as inducers or repressors) that activate, control or deactivate 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 compounds that promote organoid analysis. Exemplary reagents include markers, fixatives, and clearing agents, etc. Markers may include dyes (e.g., visible colorants and / or photoluminescent dyes). Photoluminescent dyes are any substances that emit light in response to the irradiation of excitation light.

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

[0079] Each reservoir 64a, 64b can have an open top 72, so that fluid transfer device (such as pipette or other pump) is introduced and removed.Container 50 can include a single removable lid 74, and lid 74 is matched on the container to cover the open top 72 of each reservoir 64a, 64b during cultivation in an incubator. Alternatively, the container can include two or more lids, which can be removed independently of each other and cover all reservoirs together.Each lid 74 can have a flange 76, which is configured to overlap vertically with the upper end of each reservoir, and when covering the reservoir, limits the lateral movement of the lid, optionally does not produce a tight fit.In certain embodiments, the lid can be a cap-shaped member (such as, referring to Example 8) that forms a fluid-tight seal at the top of one or more reservoirs.

[0080] Figure 2 Shows Figure 1 Exploded view of a container 50 of FIG. 5 , wherein the container is empty. The container 50 may include a lid 74, a container body 78, and a sealing member 80. The container body 78 may define reservoirs 64a, 64b and a receptacle 82 below the reservoirs. The receptacle 82 provides the top wall 58 and the transverse wall 60 of the chamber 54. However, the receptacle 82 may have an open bottom side 84, which may be sealed with a flat sealing member 80 to form the chamber 54 after a scaffold 86 supporting an organoid formation has been arranged in the receptacle 82 (see FIG. 5 ). Figure 1 82 (and Section II). A sealing member may be bonded to the bottom end of the receptacle 82 (and / or the container body 78) to form the chamber 54. In other embodiments, the sealing member may be formed in situ to seal the bottom side 84 of the receptacle 82 (see Section II).

[0081] The container 50 may have one or more optical windows to facilitate imaging (see Figure 1 ). Each optical window can be used to transmit 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 lateral 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 lateral window 90, and the lateral windows can be formed by opposing lateral walls 60 of the chamber 54. (The term "light" as used herein refers to optical radiation, including ultraviolet radiation, visible radiation (i.e., visible light), and / or infrared radiation.)

[0082] The parts of container 50 can be formed by any suitable material by any suitable procedure. In an exemplary embodiment, container body 78 can be formed by polymer (such as transparent polymer, etc.). Container body may not have removable / movable parts and / or may be formed as a single piece such as by injection molding, so that all structures (e.g., compartments) of container body are formed integrally with each other. Therefore, receiver 82 (and / or chamber 54) and reservoir 64a, 64b can have a fixed position relative to each other and / or can be non-removably / firmly attached to each other. Sealing member 80 can be formed by glass or polymer, etc., and can be preformed or formed in situ at least partially or completely inside receiver 82.

[0083] The chamber 54 (and / or the receptacle 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 size of the chamber can be designed to accommodate organoids of any suitable size (e.g., maximum 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 the volume of the chamber 54 (and / or the receptacle 82), or is about 2, 5 or 10 times the volume of the chamber 54 (and / or the receptacle 82), such as having a volume of at least about 0.5, 1, 2, 4 or 6 mL, etc.

[0084] The container 50 and / or any of its compartments may have any suitable geometric shape. For example, each of the receptacle 82, chamber 54, and / or each reservoir (e.g., reservoirs 64a, 64b) independently may have a cross-section (e.g., in a horizontal plane) that is polygonal (e.g., rectangular), elliptical (e.g., circular), oval, rosette-shaped, or other shaped. Thus, the receptacle 82, chamber 54, and / or each reservoir may independently be cylindrical, truncated conical, rectangular prism, tapered prism, or combinations thereof, etc.

[0085] Other aspects of the container 50 that may be suitable for organoid formation, culturing, 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 containers of the present disclosure; see Figures 3 to 15 The method steps described in this section can be performed in any suitable order and combination using any of the containers, container features, and / or procedures described elsewhere herein.

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

[0089] Figure 3 The container body 78 is shown selected and placed in an inverted orientation, wherein the receptacle 82 is open at the top. In other words, the top wall 58 is located below the open bottom side 84. This orientation uses gravity to promote fluid transfer to the top wall 58, while the lateral wall 60 prevents fluid from flowing laterally out of the receptacle 82.

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

[0091] The hydrogel 92 may or may not be a thermoreversible gel. The hydrogel may have a gel point (gelling temperature) above the organoid culture temperature so that the scaffold of the hydrogel is formed by cooling as the scaffold is printed. Alternatively or additionally, the polymerization reaction to form the hydrogel may be photoinduced using optical radiation such as ultraviolet light or visible light.

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

[0093] During the formation of the support 86 in the hydrogel 92, one or more channels 68a, 68b can be extended by 3D printing to supply nutrients and / or effectors to the hydrogel 92 and the cells 96 therein during the formation and cultivation of the organoid. The resulting channel extension portion can be one or more transverse permeable tubes embedded in the hydrogel 92, and can branch to form a channel network in the hydrogel. The channel network and / or the transverse permeable tube embedded in the hydrogel 92 can extend between at least one pair of channels 68a, 68b of the container body 78. Effectors (such as differentiation factors) can be supplied via the channel network to establish a concentration gradient in the hydrogel for more controlled stem cell differentiation and organoid formation. These tubes can be formed by light-induced polymerization or thermal polymerization, etc. Preformed tubes can also or alternatively be incorporated into the hydrogel or support for additional promotion of liquid flow in the hydrogel / support.

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

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

[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 reservoirs via the chambers. Figure 7In the embodiment of the present invention, the top of the culture medium 66a in reservoir 64a is higher than the top of the culture medium 66b in reservoir 64b. Therefore, gravity drives the culture medium 66a to flow 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, 66b will tend to be equal. Therefore, in order to drive further flow of fluid in the same or opposite directions, fluid can be added to one or more reservoirs and / or removed from one or more reservoirs, or the vertical direction of container 50 can be periodically changed (e.g., see Example 2).

[0097] Figure 8 and Fig. 9 A container 50 containing an organoid 52 is shown. The organoid is formed by proliferation and differentiation of cells 96 in the hydrogel 92. Fig. 9 As scaffold 86 is remodeled by organoid cells, the size and shape of organoid 52 changes relative to Figure 8 In other embodiments, the final size and shape of organoid 52 can be substantially defined by the size and shape of scaffold 86 when it is formed.

[0098] Fig.10 An exemplary imaging system 100 for capturing an image 102 of an organoid 52 by light sheet microscopy is shown. The imaging system may include an illumination assembly 104 including a light source 106 that produces light for a light sheet 108. Fig.10 As shown, the light sheet can be oriented horizontally and can propagate through the organoid 52 from one of the lateral windows 90. Alternatively, the light sheet 108 can be oriented vertically and can propagate vertically through the organoid 52 from the bottom window 88. Light can be collected from the organoid 52 using an objective lens 110 for capture by an 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 lateral 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 to provide three-dimensional image data of the organoid. If the organoid 52 no longer needs to be alive, the organoid can be fixed and cleared prior to imaging by providing fixation and clearing agents from one or more reservoirs 64a, 64b through the top wall 58 of the chamber 54.

[0099] Figures 11 to 13 An alternative in-situ method of sealing the open side 84 of the receptacle 82 to form the chamber 54 is shown. Fig.11The inverted container body 78 is shown after forming the hydrogel 92 including the scaffold 86. The filler hydrogel 113 has also been dispensed into the open receptacle 82 via the open side 84, which can prevent the receptacle 82 from leaking through the channel 68a. The filler hydrogel can be configured to melt or dissolve once the organoid culture begins. Fig.12 The 3D printer 94 is shown dispensing the sealing liquid 114 into the receptacle 82. Fig.13 Sealing liquid 114 is shown solidifying to hermetically seal the open side of receptacle 82 to form chamber 54 .

[0100] The sealing liquid can be configured to solidify 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 would be 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 melting temperature of the polymer to form a sealing layer 116 that hardens as the layer cools, thereby producing a sealing member 80.

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

[0102] Fig.14 The container body 78 is shown flipped to its organoid culture orientation, and the culture medium 56, 66a, 66b in the chamber 54 and reservoirs 64a, 64b, with the sealing layer 116 cured to form the sealing member 80. The objective lens 110 of the imaging system collects light from the chamber 54 to form an image. The culture medium 56 and the sealing layer 116 can have substantially the same refractive index to improve image quality.

[0103] Fig.15 An improved in-situ method for sealing the open side of the receptacle 82 of the container body 78 is shown (with Fig.13 Before adding the layer 116 of thermosetting sealing liquid to the receptacle 82, a radiation barrier layer 118 may be formed on the surface of the filler hydrogel 113. The layer 116 may be illuminated with light radiation from a light source 120 located above the container body 78 to promote the curing of the layer 116 to form the sealing member 80. The radiation barrier layer 118 protects the hydrogel 92 from light damage.

[0104] III. Example

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

[0106] Example 1. Strips of organoid culture

[0107] See also Fig.16 , this example describes an exemplary strip 130 for forming, culturing, monitoring and / or analyzing an array of organoids.

[0108] The strip 130 may include an array of container bodies 78 connected to one another. The container bodies may be formed integrally with one another, or may be formed separately and then connected to one another after formation (and / or after the receptacle 82 is sealed to form the 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 containers 82 in a linear shape or at least a two-dimensional array.

[0109] Example 2. Transferring fluids between containers and reservoirs

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

[0111] Fig.17 Strip 130 is shown during the culture of organoid 52 in chamber 54. Each container 50 has an independent flow cycle indicated by arrows 132, 134. Due to the level difference of culture medium 66a, 66b in corresponding reservoir 64a, 64b, flow 132 through each chamber 54 between channels 68b and 68a is driven by gravity. Supplementary flow 134 from reservoir 64a to reservoir 64b is driven by pump 136. The flow rate of pump 136 can be adjusted to keep the corresponding level of culture medium 66a, 66b substantially constant so that the fluid passes through chamber 54 at a substantially constant rate. The flow can exert pressure on organoid 52, which can promote the growth and development of organoid. In other examples, the flow rate of pump 136 can be varied, thereby exerting varying pressure on organoid 52.

[0112] In other embodiments, pump 136 may be eliminated, such as Fig.17A and Fig. 17B130. The strip 130 can be shaken to tilt the holders from the vertical at an appropriate rate so as to periodically reverse the direction of the gravity-driven flow 132. In other words, the holders can be tilted in one direction to drive fluid from the reservoir 64a to the reservoir 64b within each container 50 via the channels 68a, 68b, and then tilted in the opposite rotational direction to drive fluid from the reservoir 64b to the reservoir 64a within each container 50 via the channels 68a, 68b.

[0113] Fig.18 Another strip 140 is shown during the culture of organoid 52 in chamber 54. Strip 140 is similar to Fig.16 and Fig.17 The strips 130 are shaped like a strip 130, wherein flow 132 is driven between the channels 68a, 68b of each container 50. However, corresponding channels 142 provide fluid communication between each pair of adjacent containers 50. More specifically, as shown by flow arrows 144, each channel 142 allows fluid to flow directly from the reservoir 64a of one container 50 to the reservoir 64b of the adjacent container 50, and vice versa. In order to maintain fluid circulation, the strips 140 can be periodically tilted (such as Fig.18 The strip 140 is tilted (as shown) to drive fluid flow from left to right toward one end of the holder, and then tilted in the opposite direction of rotation to drive fluid flow from right to left toward the opposite end of the holder. The pump 146 can transfer culture medium between the reservoirs 64a, 64b located at opposite ends of the strip 140, as represented by the flow arrows at 148. The pump can be used instead of or in addition to periodically tilting the strip 140.

[0114] Example 3. Container Example

[0115] This example describes an exemplary embodiment 150 of the container 50 of Section I having a container body 78 formed by injection molding and defining four reservoirs 64a to 64d; see Figures 19 to 25 .

[0116] Each reservoir 64a-64d may be in fluid communication with the receptacle 82 (and / or chamber 54) via a respective channel 68a-68d extending through the top wall 58 of the receptacle 82 (and / or chamber 54) (see Fig.23 and Fig.25 The upper end of each channel may be flush with the top wall 58 (see Fig.25 ).

[0117] Various optical windows may be incorporated into the container 150. After the sealing member has been bonded to the bottom end surface 152 of the container body 78 (see Fig.21) thereafter, a planar sealing member 80 (e.g., similar to a microscope cover) may provide a bottom window 88 of the chamber 54. The container body may include a pair of lateral windows 90 (see Figures 19 to 21 ), the pair of transverse windows may be slightly tilted from the vertical direction to facilitate manufacture by injection molding (see Fig.24 ).

[0118] Example 4. Container with tube

[0119] This example describes an exemplary container having a preformed and / or in-situ formed tube; see Fig.26 and Fig. 27 .

[0120] Fig.26 Another embodiment 160 of the container 50 is shown for use with Fig.25 150. The channels 68a to 68d of the container 160 are different from those of the container 150. More specifically, the channels 68b and 68c are defined in part by an annular protrusion 162 that protrudes from the bottom side of the top wall 58 of the receptacle 82 (and / or chamber 54). The lumen of any or all of the channels may be formed at least in part by the annular protrusion.

[0121] Fig. 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 can be branched or not branched. In the depicted embodiment, the tubular extension 164 connects the channels 68b and 68c to each other. In other embodiments, a porous tubular network can be formed that can connect any suitable subset or all of the channels to each other. Each tubular extension 164 can be supported by a hydrogel 92 and / or embedded in a hydrogel (see Section II). The tubular extension can have the same composition as the hydrogel, or can have a composition different from the hydrogel, for example, to minimize / promote cell attachment and / or remodeling, etc.

[0122] Example 5. Container with electromagnet

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

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

[0125] Fig.29 and Fig.30 1 shows how the electromagnet 172 can be used to attract the ferromagnetically labeled cells 174 to the hydrogel 92 and / or the scaffold 86 within the chamber 54. The scaffold 86 and the hydrogel 92 can be formed as described above. Before the receptacle is sealed to form the chamber 54, or after sealing, the ferromagnetically labeled cells 174 can be introduced into the receptacle along with the culture medium 56 from one or more overlying reservoirs through one or more channels 68a to 68d. In any case, as Fig.29 As shown in FIG, ferromagnetically labeled cells 174 are initially outside of the hydrogel 92 and are allowed to settle to the bottom chamber 54 before the electromagnet is turned on. Fig.30 It is shown how energizing the electromagnet can attract ferromagnetically labeled cells 174 to and / or into the support 86. Prior to introducing the cells into the receptacle / chamber, the ferromagnetically labeled cells can be combined with a ferromagnetic material (e.g., a compound containing iron, cobalt, and / or nickel, etc.). For example, ferromagnetic particles (such as iron oxide particles) can be provided to the cells to render the cells ferromagnetic, or the cells can be coated with a ferromagnetic probe, etc.

[0126] Example 6. Racks for container modules

[0127] This example depicts exemplary racks 180, 182 for holding containers 150 in a linear array or a two-dimensional array, respectively, to form a strip 130; see Figure 31 to Figure 34 .

[0128] Each shelf 180, 182 (interchangeably referred to as a retainer) has a series of openings 184 to accommodate the container body 78 of the container 150. The openings 184 can be arranged along the same line (shelf 180) or in a rectangular grid pattern (shelf 182), etc. Each container body 78 can be removably placed in the opening 184. The size and shape of the opening can be designed to prevent the container body from completely passing through the opening. The container body can be connected to the opening 184 by any suitable mechanism, including snap fit or a separate retainer, etc.

[0129] Example 7. Container with access tube

[0130] This example describes an exemplary container including an access tube, and the use of the access tube for housing various instruments; see Figure 35 to Figure 51 .

[0131] Fig.35 An embodiment 190 of the container 50 is shown that includes a container body 78 that is sealed at its bottom end with a sealing member 80 to form a chamber 54. The container body 78 is similar to the container body described above for the container 160 (see FIG. 1 ), except that the container body defines at least one inlet tube 192. Fig.26 ). The entry tube is open at its top end 194, but the entry tube may have a bottom end closed by a barrier 196. The barrier may be a wall region of the top wall 58 of the chamber that is shared between the entry tube 192 and the chamber 54, and the wall region prevents fluid communication between the entry tube and the chamber when the barrier is intact. To simplify the drawings, the chamber 54 and the reservoirs 64a, 64b are not shown as containing fluid (e.g., culture medium or other liquid) in any of the drawings of Example 7.

[0132] As further described below, barrier 196 may be configured to be breached by a sharp or blunt instrument to gain access to chamber 54. Fig.35 As shown, the barrier may be thinner than adjacent areas of the top wall 58, and / or the barrier may include predetermined structures (eg, predetermined frangible areas of less thickness) where the barrier may be preferentially torn or ruptured with a suitable tool.

[0133] Fig.35 An exemplary instrument 198 is shown inserted into the access tube 192. Here, the instrument 198 is a permanent magnet 200, the pole 202 of which is adjacent to the barrier 196. In other embodiments, the magnet can be an electromagnet or any other instrument disclosed herein. The magnet 200 can be strong enough to form a magnetic field that extends into the chamber 54 when the barrier 196 is intact. In other cases, the bottom end of the magnet 200 can be inserted into the chamber 54 after the barrier 196 is broken (and even the magnet can break the barrier). Whether or not the magnet enters the chamber 54, the magnet can exert an attractive force on ferromagnetic items (e.g., ferromagnetically labeled cells) within the chamber 54. The magnet can be retractable / removable and / or can be securely attached to the access tube 192.

[0134] Fig.36 Container 190 is shown, in which organoid 52 is present in chamber 54 and magnet 200 is replaced by instrument 198 configured as needle 204 (similar to Fig.35). The sharp tip 206 of the needle 204 has pierced the barrier 196 (now referred to as the ruptured barrier 196') and has entered the organoid 52. The needle can be hollow or solid. In either case, the needle can allow a sample of the organoid 52 to be collected and removed from the chamber 54 via the access tube 192 for a biopsy of the organoid. Alternatively, or in addition, the needle can be used to introduce fluids, cells, and / or effectors, etc., into the organoid 52 and chamber 54. For example, the needle can allow tissue or cells to be transplanted into the organoid to achieve a xenotransplantation, an allotransplantation, or a homologous transplantation.

[0135] Fig.37 It shows that Fig.36 190 in the container 190, except that the instrument 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 the ruptured barrier 196') and entered the organoid 52. In other embodiments, the barrier 196 can first be ruptured by a different instrument 198, such as a dedicated rupture instrument, and then replaced by the sensor / electrode 208 (or any other instrument disclosed herein, etc.). The sensor / electrode 208 can include at least one electrode or an array of electrodes for electrical stimulation of the organoid 52. Alternatively, or in addition, the sensor / electrode 208 can include any suitable sensor(s), such as an electrical sensor (e.g., a capacitive sensor), a pH sensor that measures pH, an electrochemical sensor, an oxygen sensor, a CO2 sensor, and / or the like.

[0136] Figures 38 to 40 1 and 2 show an instrument 198 configured as a piercing device 214 before, during, and after the barrier 196 at the bottom end of the access tube 192 has been pierced. Fig.38 , the tip 216 of the disrupting instrument 214 travels downward (as indicated by the motion arrow at 218) and almost reaches the barrier 196. Fig.39 In FIG. 1 , the tip 216 contacts the barrier 196 and applies a deforming force to the barrier. Fig.40 , the tip 216 has completely penetrated the barrier and entered the chamber 54. The wall region forming the barrier 196 can be sufficiently resilient to maintain radial contact with the rupture device 214 after the barrier has been pierced. This radial contact can form a fluid tight seal 220 between the container body 78 (at the ruptured barrier 216') and the rupture device 214, which can substantially prevent fluid in the chamber 54 from traveling upward into the entry tube 192.

[0137] The container 190 may have only one inlet tube 192 or may have multiple inlet tubes 192. For example, Fig.41 It shows roughly Fig.24The container 190 is cut so that three access tubes 192 are visible. As described above, each access tube 192 can terminate at a corresponding barrier 196. In addition, each barrier 196 can be a wall area of ​​the top wall 58 located between the corresponding access tube 192 and the chamber 54 and / or a wall area shared between the access tubes 192 and the chamber 54. Thus, the wall area can be a common wall area or may not be a common wall area. In the depicted embodiment, a corresponding rupture instrument 214 is provided in each of the three access tubes 192, wherein only one rupture instrument extends into the chamber 54. (The ruptured barrier is indicated by 196'.) In other embodiments, any suitable number of barriers 196 can be ruptured to provide access to the chamber 54 for any suitable combination of instruments 198.

[0138] Figure 42 to Figure 44 It shows that Figures 38 to 40 Partial cross-sectional views of various embodiments 230 of a container 50 taken before, during, and after rupturing the barrier 196 using the rupturing device 214. The barrier 196 of the container 230 has a frangible web 232 where the barrier is preferentially torn in response to pressure applied by the rupturing device 214. Fig.44 , the end of the rupturing device 214 has entered the chamber 54. Fluid may travel upward from the chamber 54 into the access tube 192, or the fluid travel may be limited by a tight radial fit of the rupturing device 214 with the access tube 192 and / or an airtight seal around the rupturing device at the top of the access tube 192, etc.

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

[0140] Fig.46 A container 190 is shown with a fiber optic imaging probe 244 disposed in an entry tube 192 and extending into the organoid 52 in the chamber 54. The imaging probe 244 can have an endoscopic front lens 246 and a fiber optic bundle 248 for transmitting light from the front lens 246 to an image sensor. The imaging probe can allow for endoscopic imaging, confocal imaging, and / or illumination only (e.g., for imaging with an image sensor not coupled to the fiber optic bundle 248, for optogenetics, etc.), etc.

[0141] Fig.47 A container 190 is shown with an illumination device 250 disposed in an entry tube 192 and extending into an organoid 52 in a chamber 54. The illumination device 250 may include an optical fiber 252 to direct light from a light source 254 to an exit aperture 256 at a 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 that is not coupled to the optical fiber 252, as a light source for wavefront sensing (adaptive optics), as a light source for optogenetics, etc.

[0142] Fig.48 A container 190 is shown with a pneumatic device 258 located in an access tube 192. The pneumatic device 258 has an inflatable balloon 260 located within the organoid 52. The balloon is operably connected to a pressure regulating device (such as a pump 262), which can inflate the balloon 260 to create internal mechanical strain in the organoid 52, as shown at 264. The pneumatic device 258 can inflate and deflate the balloon 260 to expand and contract the balloon as needed. The balloon 260 can be printed with the scaffold 86 and then connected to the tube 266 of the pneumatic device 258. In other embodiments, the balloon 260 can be introduced into the organoid 52 via a needle or other sharp object.

[0143] Fig.49 It shows that Fig.41 190, but also having a pair of pneumatic devices 258a, 258b disposed in respective access tubes 192. Each pneumatic device is similar to the pneumatic device 190, except that it has an inflatable balloon 260 located outside the organoid to generate external mechanical strain on opposite sides of the organoid 52. Fig.48 The pneumatic device is similar.

[0144] Fig.50 and Fig.51 It shows that Fig.35 and Fig.36 The container 190 is similar, but also has a pair of mounting magnets 270a, 270b extending into the chamber 54 via corresponding access tubes 192. The magnets 270a, 270b magnetically attract corresponding magnets 272a, 272b that have been attached to a prefabricated support structure 274 or biochip via magnetic attraction.

[0145] Example 8. Container with flexible membrane

[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 Fig.52 and Fig.53 .

[0147] The container 190 includes a cap 282 mounted on the top of the container body 78. The cap 282 forms an airtight seal with the container body 78. The cap includes a frame 284 that fits tightly over the top edge of the container body 78. At least one flexible membrane 286 is mounted to the frame 284 and covers at least two reservoirs (e.g., 64a, 64b). The undeformed configuration of the flexible membrane is shown in dashed lines at 288. Pressure 290 can be applied to the flexible membrane 286 above the reservoir 64a or 64b to push the membrane downward, as shown at 292. The pressure drives the fluid from one reservoir to the other via the chamber 54, as shown by arrows 294, 296. The flexible membrane 286 can responsively deflect upward, as shown at 298. As shown in FIG. Fig.52 and Fig.53 As shown, pressure may 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 the present disclosure as a series of indexed paragraphs.

[0150] Paragraph A1. A method of organoid culture and / or analysis, the method comprising: (a) sealing an open side of a receptacle to form a chamber; and (b) forming an organoid in the chamber.

[0151] Paragraph A2. The method of Paragraph A1, wherein sealing includes attaching a sealing member to an open side of the receptacle.

[0152] Paragraph A3. The method of Paragraph A2, wherein attaching the sealing member includes bonding the sealing member to a receptacle.

[0153] Paragraph A4. The method of Paragraph A3, wherein bonding includes bonding a preformed sealing member to the receptacle.

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

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

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

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

[0158] Paragraph A9. A method according to paragraph A7, wherein the receptor comprises a scaffold to promote organoid formation, and further comprises a light blocking layer located between the thermosetting resin layer and the scaffold, and wherein irradiation is performed so that light propagates through the thermosetting resin layer to the light blocking layer, and the light blocking layer substantially shields the scaffold from light.

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

[0160] Paragraph A11. The method of Paragraph A10, wherein the hydrogel comprises a scaffold that supports organoid formation.

[0161] Paragraph A12. The method of 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. A method according to any one of paragraphs A1 to A3 and A5, wherein the receiver comprises a scaffold to promote organoid formation, and wherein the 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 open side of the receiver.

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

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

[0165] Paragraph A16. A method according to paragraph A15, wherein introducing includes passing the 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 of paragraph A15 or A16, wherein introducing comprises feeding the organoid by passing nutrients through a top wall of the chamber.

[0167] Paragraph A18. The method of any of paragraphs A15 to A17, wherein introducing comprises passing a marker through a top wall of the chamber to label at least a portion of the organoid.

[0168] Paragraph A19. The method of any of paragraphs A15 to A18, wherein introducing comprises:

[0169] (i) allowing one or more effectors to pass through the top wall of the chamber, wherein the one or more effectors are selected from the group consisting of differentiation factors, growth factors, anti-cancer compounds, expression vectors, viruses, oligonucleotides, messenger RNA and small interfering RNA; and / or

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

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

[0172] The molecules of (ii) or (iii) above may include guide RNA, effector molecules expressed by CRISPR-Cas, 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 of paragraph A15 or A19, wherein introducing comprises passing one or more fixatives and / or clearing agents through a top wall of the chamber.

[0174] Paragraph A21. The method of any of paragraphs A15 to A20, wherein introducing comprises utilizing gravity to drive the fluid from the reservoir into the chamber.

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

[0176] Paragraph A23. A method according to any one of paragraphs A15 to A22, wherein the chamber is in independent fluid communication with a first overlying reservoir and a 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. A method according to paragraph A23, wherein the organoid defines an interior space located within the organoid and an exterior space located outside the organoid and within the chamber, and wherein fluid held by a first overlying reservoir is supplied to the interior space and fluid held by a second overlying reservoir is supplied to the exterior space.

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

[0179] Paragraph A26. The method of paragraph A25, wherein the pump transfers fluid from the second reservoir to the first reservoir at a rate that substantially matches the rate of the gravity-driven flow.

[0180] Paragraph A27. A 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 flow from the first reservoir to the second reservoir and from the second reservoir to the first reservoir.

[0181] Paragraph A28. A 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 channels and second channels, and wherein the first reservoir and the second reservoir are in direct fluid communication with each other via a third channel above the top wall of the chamber and separated from the top wall of the chamber.

[0182] Paragraph A29. A 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 the fluid to flow 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 of any of paragraphs A1 to A30, further comprising placing a scaffold within the receptacle prior to sealing the open side of the receptacle, the scaffold being configured to promote organoid formation.

[0185] Paragraph A32. The method of paragraph A31, wherein providing the support comprises forming the support within the receptacle.

[0186] Paragraph A33. The method of paragraph A31, wherein positioning the support comprises placing a prefabricated support in a receptacle.

[0187] Paragraph A34. The method of any of paragraphs A31 to A33, further comprising introducing biological cells into the receptacle before sealing the open side of the receptacle.

[0188] Paragraph A35. The method of paragraph A34, wherein the biological cells include stem cells.

[0189] Paragraph A36. The method of paragraph A34 or A35, wherein biological cells are introduced into the scaffold when the scaffold is formed.

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

[0191] Paragraph A38. The method of paragraph A37, wherein forming the scaffold comprises 3D printing at least two different hydrogels in a receptacle.

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

[0193] Paragraph A40. The method of any of paragraphs A31 to A39, wherein the receptacle has a wall opposite the open side, and wherein the bracket is attached to the wall.

[0194] Paragraph A41. The method of any of paragraphs A31 to A40, further comprising introducing biological cells for forming an organoid into the chamber after sealing the open side of the receptacle to form the chamber.

[0195] Paragraph A42. The method of any 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. A method according to any one of paragraphs A31 to A42, wherein the chamber is in fluid communication with a plurality of overlying reservoirs via a channel, the method further comprising forming extensions of one or more channels by 3D printing, and optionally embedding the extensions in the hydrogel.

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

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

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

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

[0201] Paragraph A48. The method of paragraph A47, wherein collecting data comprises capturing an image of at least a portion of the organoid.

[0202] Paragraph A49. The method of paragraph A48, wherein capturing comprises capturing an image by light sheet microscopy.

[0203] Paragraph A50. The method of paragraph A48 or A49, wherein capturing comprises detecting photoluminescence from the organoid.

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

[0205] Paragraph A52. A method according to paragraph A51, wherein capturing includes illuminating at least a portion of the organoid through a lateral window of the chamber and detecting optical radiation that propagates out of the chamber through a bottom window of the chamber, and vice versa.

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

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

[0208] Paragraph A55. A method according to any of paragraphs A47 to A54, wherein collecting data 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 of paragraph A55, wherein collecting data comprises forming an opening in a top wall of the chamber and removing cells and / or fluid from the chamber via the opening.

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

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

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

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

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

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

[0216] Paragraph A63. The method of any of paragraphs A60 to A62, wherein the common wall defines the following feature: the common wall is configured to be torn by mechanical pressure applied to the common wall via an instrument.

[0217] Paragraph A64. A method according to any of paragraphs A57 to A63, wherein the instrument is selected from the following group, the group comprising: a needle, a light guide operably connected to a light source, an endoscope, an electrode, an ATR probe, a pneumatic / hydraulic source optionally connected to the balloon, and a magnet.

[0218] Paragraph A65. A method according to any of paragraphs A57 to A64, wherein the instrument includes a sensor located at an end thereof.

[0219] Paragraph A66. The method of Paragraph A65, further comprising sensing parameters of the chamber and / or organoid using a sensor.

[0220] Paragraph A67. A method as described in any of paragraphs A57 to A66, wherein the device includes electrodes configured to electrically stimulate the organoid.

[0221] Paragraph A68. The method of paragraph A67, further comprising electrically stimulating the organoid using electrodes.

[0222] Paragraph A69. A method as described in any of paragraphs A57 to A68, wherein the apparatus includes a light guide optically coupled to the light source and having a hole at an end introduced into the chamber.

[0223] Paragraph A70. A method according to any of paragraphs A57 to A69, wherein the instrument includes a magnet located at the end introduced into the chamber.

[0224] Paragraph A71. A method as described in any of paragraphs A57 to A70, wherein the instrument includes an attenuated total reflectance (ATR) probe.

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

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

[0227] Paragraph A74. The method of any of paragraphs A1 to A73, further comprising introducing a test compound into the chamber.

[0228] Paragraph A75. The method of any of paragraphs A1 to A74, wherein the method comprises forming a plurality of organoids in a corresponding plurality of chambers.

[0229] Paragraph A76. The method of Paragraph A75, further comprising applying a different treatment to each organoid.

[0230] Paragraph A77. The method of paragraph A76, wherein applying the different treatments comprises introducing a different test compound into each chamber of the plurality of chambers.

[0231] Paragraph A78. The method of Paragraph A77, wherein each different test compound is a potential anti-cancer drug.

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

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

[0234] Paragraph A81. The method of any of paragraphs A76 to A80, further comprising collecting data for each organoid in the plurality of organoids to test the effects of different treatments on the organoid.

[0235] Paragraph A82. The method of paragraph A81, wherein collecting data comprises imaging each organoid in situ in its respective chamber.

[0236] Paragraph A83. The method of paragraph A81 or A82, wherein collecting data comprises analyzing a corresponding fluid associated with each organoid for an analyte.

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

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

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

[0240] Paragraph B1. A method for organoid culture and / or analysis, the method comprising: (a) forming an organoid in a chamber, wherein an access tube is above the chamber and wherein a 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 of organoid culture and / or analysis, the method comprising: (a) sealing an open side of a receptacle to form a chamber; (b) forming an organoid within the chamber; (c) supplying a substance / fluid to the chamber; and (d) capturing an image of at least a portion of the organoid while the organoid is still enclosed by the walls of the chamber.

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

[0243] Paragraph D2. The device of Paragraph D1 further comprising a support for attaching the biological cell to the wall of the receptacle.

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

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

[0246] Paragraph D5. An apparatus as described in paragraph D4, wherein the biological cells include stem cells.

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

[0248] Paragraph D7. A device as described in Paragraph D6, wherein the deposit is capable of opening by light-induced release to release the drug from the deposit.

[0249] Paragraph D8. The device of any of paragraphs D1 to D7, wherein the receptacle and the at least two reservoirs are integrally formed with one another.

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

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

[0252] Paragraph D11. A device as described in any of paragraphs D1 to D10, wherein each channel extends through a wall located between the corresponding reservoir and the receptacle, and the wall is optionally shared between the corresponding reservoir and the receptacle.

[0253] Paragraph D12. The device of any of paragraphs D1 to D11, further comprising a removable cover configured to cover an open top of each of the at least two reservoirs.

[0254] Paragraph D13. The device of any of paragraphs D1 to D12, wherein the device is in a sterile condition.

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

[0256] Paragraph D15. The device of paragraph D14, wherein an organoid is contained in the chamber.

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

[0258] Paragraph D17. The device of any 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 contained in the chamber.

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

[0260] Paragraph D19. The device of any of paragraphs D1 to D18, wherein the body defines an entry 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 device of any of paragraphs D1 to D19, further comprising a linear array of substantially identical cells connected to each other, wherein one of the cells comprises a receptor and at least two reservoirs.

[0262] As used in this disclosure, the term “exemplary” means “illustrative” or “serving as an example.” Similarly, the term “exemplary” means “to illustrate by giving an example.” The term does not connote desirability nor superiority.

[0263] The above disclosure may include multiple different inventions with independent utility. Although each of these inventions is disclosed in its preferred form, the specific embodiments disclosed and shown here should not be considered to be restrictive, because many changes are possible. The subject matter of the present invention includes all novel and non-obvious combinations and sub-combinations of various elements, features, functions and / or characteristics disclosed herein. The attached claims specifically point out certain combinations and sub-combinations that are considered to be novel and non-obvious. Inventions implemented in other combinations and sub-combinations of features, functions, elements and / or characteristics can be claimed in applications claiming priority to this application or related applications. Whether for different inventions or for the same invention, and whether wider, narrower, equal or different than the scope of the original claim, such claims are considered to be included in the subject matter of the invention disclosed herein. In addition, unless otherwise explicitly stated, the order indicators such as the first, second or third for the identified elements are only used to distinguish elements, and do not indicate the specific position or order of these elements.

Claims

1. A method for culturing an organoid, the method comprising: Set the receiver; where: The receptacle is defined by 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; A first end of a first transverse wall of the pair of transverse walls extends from a first end of the top wall, and a first end of a second transverse wall of the pair of transverse walls extends from a second end of the top wall; and The second end of the first transverse wall of the pair of transverse walls and the second end of the second transverse wall of the pair of transverse walls do not contact each other; positioning the receptacle and a reservoir integrally formed with the receptacle in an inverted position wherein the receptacle is disposed above the reservoir; Disposing a bracket on the top wall of the receptacle; bonding a sealing member to each of a second end of a first one of the pair of transverse walls of the receptacle and a second end of a second one of the pair of transverse walls of the receptacle to form a chamber; inverting the chamber and the reservoir such that the chamber is disposed below the reservoir; and Organoids are formed in the chamber using the scaffold.

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

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

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

5. The method according to claim 1, wherein: Providing the support includes 3D printing the support in the receptacle.

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

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

8. A method for culturing an organoid, the method comprising: Set the receiver; where: The receptacle is defined by a top wall having a first end and a second end and a pair of transverse walls, the top wall having a first end and a second end, the pair of transverse walls each having a first end and a second end; A first end of a first transverse wall of the pair of transverse walls extends from a first end of the top wall, and a first end of a second transverse wall of the pair of transverse walls extends from a second end of the top wall; and The second end of the first transverse wall of the pair of transverse walls and the second end of the second transverse wall of the pair of transverse walls do not contact each other; positioning the receptacle and a reservoir integrally formed with the receptacle in an inverted position wherein the receptacle is disposed above the reservoir; placing a bracket on the top wall of the receptacle; attaching a sealing member to each of a second end of a first one of the pair of transverse walls of the receptacle and a second end of a second one of the pair of transverse walls of the receptacle to form a chamber; inverting the chamber and the reservoir such that the chamber is disposed below the reservoir; and When the sealing member is attached to each of the second ends of the first of the pair of transverse walls of the receptacle and the second of the second of the pair of transverse walls of the receptacle, an organoid is formed in the chamber using the scaffold.

9. The method according to claim 8, wherein: Providing the support includes 3D printing the support in the receptacle.

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: Attaching the sealing member includes coupling the sealing member to each of a second end of a first one of the pair of transverse walls and a second end of a second one of the pair of transverse walls of the receptacle.

12. The method according to claim 8, further comprising: forming an opening in a top wall of the chamber; as well as The end of the instrument is inserted from the opening into the chamber.

13. The method according to claim 12, wherein: The instrument is selected from the group consisting of a needle, a light guide operably connected to a light source, an endoscope, an electrode, an ATR probe, a source of pneumatic / hydraulic power coupled to a balloon, and a magnet.

14. A method for culturing an organoid, the method comprising: Set the receiver; where: The receptacle is defined by a top wall having a first end and a second end and a pair of transverse walls, the top wall having a first end and a second end, the pair of transverse walls each having a first end and a second end; A first end of a first transverse wall of the pair of transverse walls extends from a first end of the top wall, and a first end of a second transverse wall of the pair of transverse walls extends from a second end of the top wall; and The second end of the first transverse wall of the pair of transverse walls and the second end of the second transverse wall of the pair of transverse walls do not contact each other; positioning the receptacle and reservoir in an inverted position wherein the receptacle is disposed above the reservoir; placing a bracket on the top wall of the receptacle; attaching a sealing member to each of a second end of a first one of the pair of transverse walls of the receptacle and a second end of a second one of the pair of transverse walls of the receptacle to form a chamber; inverting the chamber and the reservoir so that the chamber is disposed below the reservoir; forming an organoid in the chamber using the scaffold; and Fluid and / or at least one substance is introduced from the reservoir integrally formed with the receptacle through the top wall into the chamber to contact the organoid.

15. The method according to claim 14, wherein: The introducing comprises allowing the fluid and / or at least one substance to enter the chamber via a channel extending through a wall between the reservoir and the chamber.

16. The method according to claim 14, wherein: The introducing includes passing nutrients through a top wall of the chamber to feed the organoid.

17. The method according to claim 14, wherein: The introducing includes passing one or more fixing agents and / or clearing agents through the ceiling of the chamber.

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

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

20. The method of claim 14, further comprising: forming a plurality of organoids in the corresponding plurality of chambers; as well as A different treatment is applied to each of the plurality of organoids.

21. The method according to claim 20, wherein: Applying the different treatments includes introducing a different test compound into each chamber of the plurality of chambers.

22. The method according to claim 20, wherein: Applying the different treatments involved changing the genetic material in the corresponding compartment of each organoid through genome editing.

23. The method according to claim 20, wherein: Applying the different treatments comprises using one or more DNA binding agents to mark at least one genomic locus of each organoid or alter the gene expression profile of each organoid in vivo in the corresponding compartment of each organoid.

24. The method of claim 20, further comprising collecting data from 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: Collecting the data includes imaging each organoid in situ in its corresponding chamber.

26. The method according to claim 24, wherein: Collecting the data includes analyzing the corresponding fluid 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 according to claim 27, wherein: Collecting the data includes physically sectioning each organoid after removing it from its chamber.

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

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