Systems, methods, and devices for culturing multicellular structures

Abstract

Systems, methods, and devices for culturing multicellular structures, such as organoids. Exemplary systems include a container, an electric / magnetic module, and control circuitry. The container may include a culture chamber for containing the multicellular structures. The electric / magnetic module may be configured to be located within the container, within or adjacent to the culture chamber. The control circuitry may be configured to wirelessly power and / or operate the electric / magnetic module.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 976,151, filed February 13, 2020, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Organoids ("mini-organs") are three-dimensional clusters of different cell types that are generated in vitro and bear some resemblance to organs, such as exhibiting realistic histology of organ-specific tissues. Cell clusters can be generated by seeding a small number of stem cells into a matrix. The stem cells then proliferate, differentiate, and self-organize within the matrix, using the matrix as a scaffold. This approach has previously generated organoids resembling tissues from the brain, heart, intestine, kidney, liver, and stomach, among others. These promising results suggest that organoid cultures have the potential to provide new insights into organ development and function and to recapitulate disease models, enabling in vitro drug screening. Organoids could revolutionize the way drugs are discovered and personalized.

[0003] Despite the growing importance of organoids, they remain a challenge to culture efficiently. New systems, methods, and devices for culturing organoids and other multicellular structures are needed. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides systems, methods, and devices for culturing multicellular structures, such as organoids. An exemplary system includes a container, an electric / magnetic module, and control circuitry. The container may include a culture chamber for containing the multicellular structures. The electric / magnetic module may be configured to be located within the container, within or adjacent to the culture chamber. The control circuitry may be configured to wirelessly power and / or operate the electric / magnetic module. The present invention provides, for example, the following items. (Item 1) 1. A system for culturing multicellular structures, comprising: a container comprising a culture chamber configured to contain the multicellular structure; an electric / magnetic module configured to be positioned within the vessel at a location within or adjacent to the culture chamber; a control circuit configured to wirelessly power and / or operate the electric / magnetic module; and A system comprising: (Item 2) Item 10. The system of item 1, wherein the control circuit is configured to wirelessly transfer power to the electric / magnetic module via inductive or capacitive coupling. (Item 3) 3. The system of any of items 1-2, wherein the electric / magnetic module is contained within or configured to be contained within the culture chamber and includes a magnet, and the control circuitry is configured to create a magnetic field that drives movement of the magnet within the culture chamber. (Item 4) Item 10. The system of item 1, wherein the electric / magnetic module includes a chemical sensor, an electric sensor, an optical sensor, and / or a temperature sensor. (Item 5) 5. The system of any of items 1-4, wherein the electric / magnetic module includes a pump configured to drive fluid flow into and / or out of the culture chamber. (Item 6) 6. The system of any of items 1-5, wherein the electric / magnetic module is located or configured to be located within a slot adjacent to the culture chamber, and optionally, the vessel defines an opening providing fluid communication between the slot and the culture chamber. (Item 7) 7. The system of item 6, wherein the container includes two or more reservoirs in fluid communication with the culture chamber, the slot being located vertically above the culture chamber, optionally between at least a pair of the two or more reservoirs, and optionally each reservoir of the two or more reservoirs being separately in communication with the culture chamber. (Item 8) 8. The system of any of items 1-7, further comprising a container assembly connected to one another to form a container row containing the containers, each container in the container row comprising an individual culture chamber for containing the multicellular structures, and optionally, the containers in the container row are substantially identical to one another. (Item 9) 9. The system of any of items 6-8, further comprising a frame for holding a plurality of container assemblies including the container assembly, the frame optionally having a length and / or width corresponding to the length and / or width of a standard microplate footprint. (Item 10) 1. A method for culturing a multicellular structure, comprising: containing a multicellular structure within a culture chamber of a vessel, wherein an electric / magnetic module is located within the vessel at a location within or adjacent to the culture chamber; wirelessly powering / operating said electric / magnetic module using a control circuit; A method comprising: (Item 11) Item 11. The method of item 10, wherein powering / operating includes wirelessly transmitting electrical energy to the electric / magnetic module. (Item 12) 12. The method of claim 10 or 11, wherein powering / operating is performed at least in part via inductive or capacitive coupling of the control circuit and the electric / magnetic module to each other. (Item 13) 13. The method of any of items 10-12, wherein the electric / magnetic module includes a magnet, and powering / operating includes using a magnetic field created by the control circuitry to drive movement of the magnet within the culture chamber. (Item 14) 14. The method of any of items 10-13, wherein powering / operating comprises sensing properties of the multicellular structure and / or culture medium in the culture chamber using a sensor of the electric / magnetic module. (Item 15) 15. The method of any of items 10-14, wherein powering / operating comprises driving fluid flow into and / or out of the culture chamber. (Item 16) 16. The method of any of items 10-15, further comprising collecting data associated with the multicellular structure while the multicellular structure remains within the culture chamber. (Item 17) Item 17. The method of item 16, wherein collecting data is performed using a sensor of the module. (Item 18) 1. A device for culturing multicellular structures such as organoids, said device comprising: a shell having an open top; an insert including two or more reservoirs, the insert configured to be received within the shell via the open top such that the shell and the insert cooperatively form a culture chamber for the multicellular structure, the culture chamber being located below the two or more reservoirs and in fluid communication with each of the two or more reservoirs via a separate channel defined by the insert; A device comprising: (Item 19) Item 19. The device of item 18, further comprising a scaffold configured to support organoid formation within the culture chamber. (Item 20) 20. The device of claim 19, wherein the scaffold is provided by a module that is positioned within or configured to be positioned within a slot defined by the inserter. (Item 21) 21. A device according to any of items 18-20, wherein the shell includes a section having an upper region defining a receiving space and a lower region forming a receptacle, the insert being configured to be received within the receiving space, and the receptacle being configured to cooperatively form the culture chamber together with the insert. (Item 22) 22. The device of any of items 18-21, wherein the inserter forms a slot and defines an opening at a bottom end of the slot, and further comprises a module located or configured to be located within the slot vertically above the opening, optionally the module being an electric / magnetic module. (Item 23) 1. A system for culturing multicellular structures, said system comprising: a container comprising a culture chamber for containing the multicellular structure and two or more reservoirs and slots, each reservoir and slot being in fluid communication with the culture chamber; two or more modules having different functions from one another and configured to be interchangeably installed in said slots; A system comprising: (Item 24) 24. The system of claim 23, wherein the container defines an opening at a bottom end of the slot that communicates with the culture chamber. (Item 25) 25. The system of any of items 23-24, wherein the two or more modules include a first module having a permeable membrane. [Brief explanation of the drawings]

[0005] [Figure 1]FIG. 1 is a block diagram of an exemplary culture system for forming, growing, feeding, differentiating, stimulating, sensing, testing, and / or imaging multicellular structures, the culture system including a container array for containing the multicellular structures in individual culture containers, at least one electric / magnetic module located in at least one culture container of the container array, and control circuitry for powering and / or operating the electric / magnetic module via wireless transmission between the electric / magnetic module and the control circuitry.

[0006] [Figure 1A] FIG. 1A is a schematic diagram of an exemplary culture vessel of the vessel array of FIG.

[0007] [Figure 2] FIG. 2 is an exploded schematic top view of an embodiment of the vessel array of FIG.

[0008] [Figure 3] FIG. 3 is an exploded schematic top view of another embodiment of the vessel array of FIG.

[0009] [Figure 4] FIG. 4 is a schematic diagram of the culture vessel of FIG. 1A and a set of functionally distinct modules for use within the culture vessel.

[0010] [Figure 5] FIG. 5 is a block diagram of an exemplary embodiment of the culture vessel and control circuitry for the culture system of FIG. 1, with the culture vessel shown in a schematic side view.

[0011] [Figure 6] FIG. 6 is a flow chart of exemplary steps that may be performed in a method of culturing a multicellular structure.

[0012] [Figure 7]FIG. 7 is a flow chart of exemplary steps that may be performed in a method of culturing a multicellular structure using a culture vessel that includes a shell and an insert.

[0013] [Figure 8] FIG. 8 is a somewhat schematic diagram of an exemplary electrode module for use in the vessel of FIG. 1 or the method of FIG. 6, along with the power and communication antenna of control circuitry configured to power and operate the electrode module.

[0014] [Figure 9] FIG. 9 is a somewhat schematic diagram of an exemplary sensor module for use in the container of FIG. 1 or the method of FIG. 6, along with an antenna of control circuitry configured to power and operate the sensor module.

[0015] [Figure 10] FIG. 10 is a somewhat schematic diagram of an exemplary pump module for use in the culture system of FIG. 1 or the method of FIG. 6, along with a power antenna of a control circuit configured to power and operate the pump module.

[0016] [Figure 11] Figure 11 is a partial cross-sectional, partial schematic diagram of selected aspects of an implementation of the system of Figure 1, including two copies of the pump module of Figure 10 contained within the culture chamber of a culture vessel, in a position adjacent to the organoids.

[0017] [Figure 12] Figure 12 is a partial cross-sectional, partial schematic diagram of selected aspects of an implementation of the system of Figure 1, including control circuitry, the culture vessel of Figure 11, and a pair of magnetic modules contained within the culture chamber of the culture vessel, with the organoids positioned between the magnetic modules.

[0018] [Figure 13]Figure 13 is another view of the system implementation of Figure 12 taken with the coils of the control circuit energized to drive movement of the magnetic modules toward each other to mechanically stimulate the organoids.

[0019] [Figure 14] Figure 14 is a schematic side view of the system implementation of Figure 12 modified for culturing and mechanically stimulating a set of organoids contained within a row of culture vessels, obtained with the culture vessels omitted and the coils of the control circuit not electrically energized.

[0020] [Figure 15] FIG. 15 is another schematic side view of the modified system implementation of FIG. 14 obtained with the coils electrically energized to drive linear motion of each pair of magnetic modules along a row of culture vessels.

[0021] [Figure 16] FIG. 16 is a schematic top view of a modified implementation of the system of FIG. 14 having a different configuration of electrically energizable coils for driving linear movement of the magnetic modules along the same line.

[0022] [Figure 17] FIG. 17 is a somewhat schematic diagram of an exemplary optical module for use in the culture vessel of the present disclosure.

[0023] [Figure 18] FIG. 18 is a somewhat schematic illustration of an exemplary scaffold module that includes a scaffold attached to the body of the module and configured to support organoid formation and growth.

[0024] [Figure 19] FIG. 19 is a cross-sectional view of an exemplary permeable interface module for use in the culture system of FIG. 1 or the method of FIG.

[0025] [Figure 20]FIG. 20 is a cross-sectional view of the permeable interface module of FIG. 19 operatively positioned within a slot of a culture vessel containing organoids.

[0026] [Figure 21] Figure 21 is an isometric view of a frame holding an exemplary culture device for use in the system of Figure 1 or the method of Figure 6, the culture device including container assemblies forming a row of culture vessels including corresponding rows of culture chambers, each containing a pair of magnetic modules and each located adjacent to an individual slot containing another module that is structurally and functionally different from the magnetic module.

[0027] [Figure 22] 22 is an isometric view of the culture device of FIG. 21 after removal from the frame of FIG. 21 and operative alignment of one of the culture vessels of the vessel assembly with the light source and objective of the imaging system.

[0028] [Figure 23] FIG. 23 is an isometric exploded view of the culture device of FIG. 21, including a lid not shown in FIG. 21 or FIG.

[0029] [Figure 24] FIG. 24 is a side view of the culture device of FIG. 21 taken in isolation.

[0030] [Figure 25] FIG. 25 is a top view of the culture device of FIG. 21 taken in isolation.

[0031] [Figure 26] 26 is a fragmentary, partial cross-sectional view of the culture device of FIG. 21 taken generally along line 26-26 of FIG. 25.

[0032] [Figure 27] FIG. 27 is a side view of the shell of the culture device of FIG. 21 taken in isolation.

[0033] [Figure 28] FIG. 28 is a top view of the shell of FIG.

[0034] [Figure 29] FIG. 29 is a cross-sectional view of the shell of FIG. 28 taken generally along line 29-29 of FIG. 28 through one of the four sections of the shell.

[0035] [Figure 30] 30 is another cross-sectional view of the shell of FIG. 28 taken generally along line 30-30 of FIG.

[0036] [Figure 31] FIG. 31 is a side view of the inserter of the culture device of FIG. 21 taken in isolation.

[0037] [Figure 32] 32 is an end view of the inserter of FIG. 31. FIG.

[0038] [Figure 33] 33 is a top view of the inserter of FIG. 31. FIG.

[0039] [Figure 34] 34 is a cross-sectional view of the inserter of FIG. 31 taken generally along line 34-34 of FIG.

[0040] [Figure 35] 35 is an exploded view of a lid assembly for a container array including a row of container assemblies held by the frame of FIG. 21, the lid assembly including a series of lids each substantially identical to the lid of FIG. 23.

[0041] [Figure 36] FIG. 36 is a modified form of the inserter of FIG. 31 including a gasket for creating a fluid-tight seal with the shell of FIG.

[0042] [Figure 37] 37 is a cross-sectional view of the inserter of FIG. 36 taken generally along line 37-37 of FIG.

[0043] [Figure 38] FIG. 38 is a top view of an exemplary non-slotted insert for installation into the shell of FIG. 27 to cooperatively form a culture chamber with the shell.

[0044] [Figure 39] FIG. 39 is a top view of an exemplary four-reservoir insert for installation into the shell of FIG. 27 to cooperatively form a culture chamber with the shell.

[0045] [Figure 40] FIG. 40 is a top view of an exemplary slotless four-reservoir insert for installation into the shell of FIG. 27 to cooperatively form a culture chamber with the shell. DETAILED DESCRIPTION OF THE INVENTION

[0046] Detailed Description Culturing large organoids (e.g., up to approximately 4 mm) is labor-intensive and complex, and can easily require several months before the organoids can be harvested or used (e.g., for screening). During culture, organoids pass through different stages of the culture protocol as they grow, differentiate, and develop. Currently, most of these stages require manual interaction for feeding, monitoring, handling, etc. Because the culture vessel containing the organoids may not be suitable for monitoring the state of the developing organoids, changing laboratory equipment is often necessary. Culture protocols that rely on manual interaction are expensive and prone to process errors. Furthermore, to culture large organoids that develop specific interacting cell types, it may be essential not only to treat the cells with appropriate compounds, but also to expose the organoids to a suitable physical environment specific to the type of organoid being cultured. For example, cardiomyocytes and neurons need to be stimulated by electrical pulses, while muscle and bone organoids rely on the application of alternating mechanical strain. Organoids can be cultured using complex instrumentation that relies on tubing and wiring, however, this instrumentation is bulky, non-standardized, and not easily scaled up to simultaneously culture many organoids.

[0047] In various aspects, the present disclosure addresses the lack of automation, the existence of non-standardized instruments, tubing and wiring, and non-scalability of organoid culture as currently practiced.More specifically, the present disclosure provides containers, modules, and control circuits that enable automation, improve standardization, avoid tubing and wiring, and enable scalability.

[0048] The present disclosure provides systems and methods for culturing multicellular structures, such as organoids. The system may include a container including a culture chamber for containing the multicellular structures. An electric / magnetic module may be configured to be located within the container, in or adjacent to the culture chamber. A control circuit may be configured to power and / or operate the electric / magnetic module via wireless transmission. In the method, the multicellular structures may be contained within the culture chamber of the container. The electric / magnetic module may be located within the container, in or adjacent to the culture chamber. The electric / magnetic module may be powered / operated using the control circuit via wireless transmission of power, force, and / or signals (e.g., data). The electric / magnetic module may include a magnet that can be or is moved within the container by a magnetic field created by the control circuit, for example, so that the module pumps fluid or mechanically stimulates the multicellular structures contained within the culture chamber. In other examples, the electrical / magnetic module may include electrodes for stimulating the multicellular structure, sensors for sensing properties of the multicellular structure and / or culture medium within the container, a light source for illuminating at least a portion of the multicellular structure, and / or the like.

[0049] The ability to wirelessly power / operate the electric / magnetic module in the present system and method is advantageous because it eliminates the need for wires or other electrical conductors extending from the control circuitry to the vessel. As a result, the vessel becomes more portable, with fewer restrictions on where it can be positioned relative to the control circuitry, and can be more easily separated from the control circuitry (e.g., to perform imaging procedures). This wireless approach may also improve the fidelity and stability of experiments involving multicellular structures, as it allows the electric / magnetic module to be positioned in close proximity to or in contact with the multicellular structure under analysis. The present system and method may also facilitate the automation of complex cultures of multicellular structures by providing the necessary physical environment, enabling multichannel in situ monitoring of multicellular structures. The present system and method may be capable of stimulating, maintaining, and monitoring large numbers of organoids simultaneously in an automated manner.

[0050] The container of the present disclosure may be customized as needed by introducing one or more active / passive modules into one or more compartments of the container. The selection of functional modules and the compartments into which the modules are received allows the container to be functionally adapted during manufacturing and / or by the user. For example, the selection of modules and compartments allows, among other things, the cultivation of specific types of organoids, the implementation of specific culture protocols or protocol steps, the creation of desired test conditions, and / or the on-board (in-vessel) sensing, measurement, and / or monitoring of desired parameters. Thus, while the basic structure of the container can be standardized, the functionality of the container can be modified as needed by introducing different modules to meet the specific needs of various users. In addition, the introduction of modules into the container allows the modules to be in close proximity to or in contact with the multicellular structure, which generates a more direct interaction between the modules and the multicellular structure. Furthermore, the introduction of modules into the container may not cause any increase in the size of the footprint. Thus, vessels modified with modules can remain compact, allowing more copies of the vessel to fit within the footprint of a standard microplate, which in turn allows more multicellular structures to be cultured simultaneously within the incubator.

[0051] A device and method are provided for culturing multicellular structures such as organoids. The device may comprise a shell with an open top. The device may also comprise an inserter comprising two or more reservoirs. The inserter may be configured to be received in the shell through the open top, so that the shell and the inserter cooperatively form a culture chamber for the multicellular structure. The culture chamber may be located below the two or more reservoirs and be in fluid communication with each of the two or more reservoirs through individual channels defined by the inserter. In the method, the inserter comprising two or more reservoirs may be placed in the shell, and the inserter and the shell may be used cooperatively to form a culture chamber. The culture chamber may be located below each of the two or more reservoirs and be in fluid communication with it. The multicellular structure may be cultured in the culture chamber.

[0052] The devices and methods described in the preceding paragraphs may provide various advantages for culturing multicellular structures such as organoids, including any combination of the following: The inserts may be selected from a set of inserts having different properties, such as different reservoir, channel, and / or slot configurations. Thus, the same shell may be assembled with different types of inserts to customize the resulting container structure for the user's specific needs. The inserts may also position the reservoirs (and optional slots) vertically above the culture chamber so that gravity can drive fluid flow into and / or out of the culture chamber. Furthermore, the shells may each define a row of sections capable of receiving an insert, forming individual culture chambers. Thus, the shell and two or more inserts can be assembled to form a container assembly having a row of culture chambers. Furthermore, two or more of the container assemblies may be held by a frame having a footprint corresponding to that of a standard microplate, allowing for the formation of a compact array of containers. Each container assembly may be individually removable from the frame for handling separately from the other container assemblies in the array.

[0053] The container can provide multiple reservoirs, and the reservoirs and chambers can be formed in the shared wall between the channels, and the fluid is connected to the chamber.This configuration can be described as standard feeding interface.In some embodiments, 3D printing can provide the connection of the standard feeding interface inside the container to any suitable printed structure, and can allow different types of organoids to grow.

[0054] When they develop into organoid, matrix can provide the temporary scaffold for appropriate type of cells.Cells can self-organize and generate their own extracellular matrix, which can replace part or all of scaffold.The same can be applied to internal feeding, container can provide a general interface, which can be optionally modified by 3D printing, and cells can self-organize to best use this modified interface.

[0055] In some embodiments, scaffold (with or without cells) can be placed in the receptacle of the container body, and a culture chamber can be formed from the receptacle by using a sealing member while the container is turned upside down.Once these processes are completed, the container can be turned right side up (with respect to its organoid culture orientation), and at least one reservoir above the culture chamber can be filled with feeding fluid.If there are no cells inside the scaffold yet, suitable cells can be placed in feeding fluid and introduced into the scaffold together with the feeding fluid from the reservoir above the culture chamber.

[0056] Formed organoid can require initial culture time before specific feeding protocol can be started.Feeding protocol can involve filling reservoir with suitable medium, and removing medium from reservoir according to predetermined schedule and / or based on organoid development stage or condition.Feeding protocol can depend on the shape of scaffold and the type of organoid to be formed.

[0057] The container may enable light sheet 3D imaging. The culture chamber of the container may have two, three, or more optical windows, and light may propagate into and / or out of the culture chamber through each optical window. For example, the container may have a bottom window and one or more side windows, each of which may be planar. In some embodiments, the container may have a pair of side optical windows arranged opposite each other.

[0058] This disclosure enables the generation of large, functional organoids. Large organoids may exceed approximately 0.1, 0.2, 0.5, 1, or 2 millimeters in average or maximum diameter, among others. Working with large organoids remains challenging, and researchers face two major limitations. First, each type of organoid may require different culture conditions, such as specific hydrogels as scaffolds, or even mechanical stimuli such as shear forces caused by medium flow. Second, microscopic examination of large organoids can be extremely difficult. The most current method remains thin-sectioning, staining, and imaging fixed samples using a confocal scanning microscope or even a slide reader.

[0059] The present disclosure provides improved systems, methods, and devices for organoid culture. By using a combination of 3D printing (scaffolds and / or cells) and gravity flow medium exchange, users can generate unique 3D environments optimized for each organoid type. A wide range of different organoid types can be grown. Feeding and waste removal can be handled by fluid communication between the container's culture chamber and a reservoir. Integrating optical windows into each container, at least one for the entrance of excitation light and another for the exit of emission light, allows for the monitoring of live cells in organoids by light sheet microscopy. Alternatively, or in addition, organoids can be imaged by classical wide-field microscopy through one or more of the optical windows. Thus, the containers disclosed herein can enable live cell microscopy of developing and / or developed organoids. High-content and / or high-throughput microscopy can be performed on organoids.

[0060] Further aspects of the present disclosure are described in the following sections: (I) Definitions, (II) Overview of Culture Systems and Methods, (III) Electric / Magnetic Modules, (IV) Passive Modules, (V) Vessel Assemblies, and (VI) Selected Aspects. I. Definition

[0061] Technical terms used in this disclosure have meanings commonly recognized by those of ordinary skill in the art. However, the following terms may be further defined as follows:

[0062] Cell - the basic structural, functional, and biological unit of a living organism. Cells can be eukaryotic or prokaryotic. Exemplary cells include stem cells, differentiated cells, established cell lines (e.g., cell lines), primary cells, cells of a tissue sample, transfected cells, cells from a clinical sample (e.g., a blood sample, a fluid aspirate, a tissue explant, etc.), cells that form a whole organism, and / or the like.

[0063] Any suitable cells may be introduced into the culture chamber (or into a receptacle that will form part of the culture chamber). The introduced cells may include stem cells (e.g., pluripotent stem cells), feeder cells, and / or the like. The cells may be deposited into the culture chamber or receptacle, and / or into a scaffold located or to be located within the culture chamber or receptacle, by any suitable technique, including pipetting, bioink droplet printing, microcontact printing, photolithography, dip-pen nanolithography, and / or the like.

[0064] Cell culture - promoting the survival, health, growth, proliferation, differentiation, and / or self-organization of living cells, such as cells in multicellular structures, in an artificial environment.

[0065] Culture chamber - a compartment containing multicellular structures and having walls on substantially all sides of a mostly or completely enclosed space, at least one of the walls may define one or more openings to allow communication with and / or passage into and / or out of the compartment.

[0066] Culture medium - an aqueous composition for cell culture. The composition may be liquid or semi-solid. The composition may include, among other things, a carbon source (e.g., glucose), inorganic salts, vitamins, and growth regulators. The term "medium" as used herein means at least one culture medium and may refer, for example, to separate volumes of medium, a first medium and a second medium of different composition, medium of substantially the same composition in contact with different / separate cell cultures, or a combination / mixture of previously separated volumes of the same medium.

[0067] Culture vessel—a device for culturing multicellular structures. A culture vessel (synonymously referred to as a vessel) may include a culture chamber and one or more reservoirs in fluid communication with the culture chamber. A vessel assembly or vessel array is a collection of culture vessels for culturing one-, two-, or three-dimensional arrays of multicellular structures. The culture vessels disclosed herein may be single-use devices (consumables) or may be reused.

[0068] Exemplary - To be illustrative or serve as an example. Similarly, the term "exemplify" means to illustrate by giving an example. Neither term implies desirability or superiority.

[0069] Within - When describing the location / position of an object relative to a given structure, "within" or "inside" means that the object is at least primarily (greater than 50% of the object's volume) or entirely inside the given structure. In the same context, "outside" means that the object is at least primarily (greater than 50% of the object's volume) or entirely outside the given structure.

[0070] Light - optical radiation, including ultraviolet radiation, visible radiation (i.e., visible light), and / or infrared radiation.

[0071] Module—A structurally and functionally discrete unit configured to be contained within a culture vessel. The module may be insertable into and / or removable from the culture vessel while the culture vessel is intact or only when the culture vessel is disassembled, or may be configured to be non-removable from the culture vessel. A module may be an active module, also called an electric / magnetic module, which is a module that utilizes electricity and / or magnetism for operation and, optionally, is powered / operated by wireless transmission between control circuitry and the module. Alternatively, a module may be a passive module, which is a module that does not utilize either electricity or magnetism to fulfill its intended purpose. Electric / magnetic modules include magnetic modules that comprise permanent magnets but no electric / electronic devices, electric modules that comprise electric / electronic devices but no permanent magnets, and modules that comprise permanent magnets and electric / electronic devices. A magnetic module may require an externally generated, optionally time-varying, magnetic field for operation (such as to drive the movement of the magnetic module and / or its magnet).

[0072] The modules may have any suitable shape and size. The module, and in particular its housing or body, may be, for example, cubic (e.g., cubic), cylindrical, conical, or the like. The shape of the module in cross section may correspond to that of a slot or other compartment of the container, such that the module fits into the slot or other compartment. To provide flexibility and interchangeability, it may be advantageous to have the same standard shape and size for all modules configured to be installed in a given compartment (e.g., its slot) of the container, or the same standard size and shape for all modules, regardless of the compartment's destination. 4 x 4 x 4 mm 3 are exemplary sizes of the active and passive modules, which correspond to the size of large organoids that can be generated within the culture chamber of the vessel.

[0073] Multicellular structure - a three-dimensional arrangement of interconnected biological cells. Multicellular structure may be an organized multicellular structure, which is a multicellular structure consisting of different cell types that are arranged non-randomly relative to each other. Exemplary multicellular structures include organoids, organisms (at any developmental stage), tissue explants, tumors, or the like.

[0074] Near Field Communication (NFC) - wireless communication between electronic devices using near-field radiation and inductive or capacitive coupling. Near field communication may be performed when the electronic devices are less than 50, 20, 10, or 5 centimeters from each other, among others.

[0075] Near-field radiation - electromagnetic radiation, typically radio waves (e.g., microwaves), within 10, 5, or 2 wavelengths of a source, such as within 50, 20, 10, or 5 centimeters of the source.

[0076] Organoids - Three-dimensional aggregates of different types of cells that are generated in vitro and have some resemblance to an organ, such as exhibiting realistic histology of organ-specific tissue. Cell aggregates can be generated by seeding a small number of stem cells onto a scaffold (i.e., matrix). The stem cells then proliferate, differentiate, and self-organize within the scaffold.

[0077] Receptacle - a container, optionally having open sides, such as an open top side, an open bottom side, or an open side side. A receptacle may be converted into a culture chamber, at least in part, by covering or closing the sides of the receptacle.

[0078] Scaffold—An extracellular support framework for culturing a multicellular structure. A scaffold is typically a matrix in which the cells of the multicellular structure are or will be embedded. The scaffold may be provided by one or more hydrogels. Each hydrogel may include one or more thermoplastic structural components, such as Matrigel, alginate, nanofibrillar cellulose, collagen, fibrin, and / or polyethylene glycol, among others, that cooperatively form the matrix in a temperature-dependent manner.

[0079] In some embodiments, two or more different hydrogels / matrices may be disposed within the culture chamber of the vessel. The hydrogels / matrices may differ with respect to any suitable parameter, such as melting temperature, resistance to enzymatic degradation, solubility, cell-attracting and / or cell-repelling properties, and / or the like.

[0080] Each hydrogel / matrix may comprise 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 linked to a core protein (e.g., via its serine) to form a core protein such as aggrecan, agrin, brevican, collagen type XVIII, leprecan, neurocan, perlecan, small leucine-rich proteoglycan, versican, or the like), fibrous proteins (e.g., collagen, elastin, fibronectin, laminin, etc.), and / or the like. Protease recognition sites (e.g., for scaffold metalloproteinases (MMPs)) may be incorporated into the hydrogel / matrix to allow degradation / remodeling by cells. The frequency of such sites, along with the sequence of each site, may be selected to allow a suitable amount of degradation / remodeling.

[0081] One or more growth factors may be included in the matrix as it is formed, or may be introduced into the culture medium after formation of the matrix. Exemplary growth factors that may be suitable include angiopoietin, bone morphogenetic protein (BMP), ciliary neurotrophic factor, colony-stimulating factors, 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 (alpha), vascular endothelial growth factor, and / or the like. II. Overview of Culture Systems and Methods

[0082] This section provides an overview of the culture systems and methods of the present disclosure (see Figures 1-7).

[0083] FIG. 1 shows an exemplary culture system 100 for forming, growing, differentiating, organizing, stimulating, sensing, analyzing, and / or imaging multicellular structures, such as organoids. The culture system 100 comprises a vessel array 101, which includes a set of culture vessels 102, only three of which are clearly identified in FIG. 1 . Each culture vessel 102 is configured to contain an individual multicellular structure. The vessel array 102 can be or include a linear array, a two-dimensional array (e.g., a rectangular array as shown, a hexagonal array, etc.), and / or a three-dimensional array. The vessel array 101 and / or each of its two or more discrete vessel assemblies may have a footprint corresponding to the length and / or width of a standard microplate to facilitate mechanical and fluidic handling using robotic systems designed to manipulate standard microplates. The number of culture vessels 102 in the vessel array 101 may be at least three in a first dimension and one or more (e.g., at least two, three, or more) in a second orthogonal dimension.

[0084] Each culture vessel 102 of the vessel array 101 may include two or more distinct compartments, which may or may not be in fluid communication with each other and which may or may not share one or more walls with each other (see FIG. 1A ). The culture vessel 102 has a culture chamber 103 for containing multicellular structures 104, such as organoids. At least one reservoir 105 of the vessel 102 is configured to hold a culture medium. Each reservoir 105 is arranged in fluid communication with the culture chamber 103 via at least one individual connecting channel 106. The vessel 102 may also have at least one slot 107, which may be in communication with the culture chamber 103 via an opening 108.

[0085] Each compartment of the culture vessel 102 may have any suitable size and shape. The culture chamber 103 may have a volume of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, or 1 mL. The culture chamber may be sized to contain multicellular structures, such as organoids, of any suitable size, such as multicellular structures having a diameter of at least 0.2, 0.5, 1, or 2 mm, among others. In an exemplary embodiment, each reservoir 105 of the culture vessel 102 has a volume at least 2, 5, or 10 times the volume of the culture chamber, and / or a volume greater than the culture chamber 103, such as at least 0.5, 1, 2, 4, or 6 mL, among others. The slot 107 may have a volume greater than, less than, or the same as the culture chamber 103 or one or more reservoirs 105, and / or may have the same height as each reservoir 105. Each compartment of the vessel may be rectangular, circular, oval, or the like in horizontal cross section. A rectangular cross section for each compartment of the container can be advantageous because this shape makes very efficient use of available space and allows culture containers 102 to be arranged very close to each other in the container array 101.

[0086] The culture vessel 102 may be formed from any suitable material by any suitable procedure. In an exemplary embodiment, the culture vessel may be composed of at least one polymer, which may include a transparent polymer. The culture vessel 102 may be integrally formed as a single piece, or may be formed by at least a pair of discrete components, such as a shell and insert or a body and seal member, that fit together and / or are attached to each other to form the culture chamber 103. Thus, the culture chamber 103, reservoir 105, and / or slot 107 may have fixed positions relative to each other and / or may be non-removably / rigidly attached to each other within the culture vessel 102.

[0087] The reservoir 105 of the container 102 may hold any suitable substance to be supplied to the culture chamber 103. Exemplary substances include, among others, nutrients, effectors, and reagents. Suitable nutrients include any substance for promoting the health and proliferation of cells inside the culture chamber 103, and thus the growth and development of multicellular structures such as organoids. Exemplary nutrients may include sugars (such as glucose), amino acids, proteins, nucleotides, vitamins, minerals, fatty acids, and the like. Effectors include any molecules (such as inducers or inhibitors) that activate, regulate, or inactivate a process or action (such as differentiation, protein synthesis, migration, and the like). Exemplary effectors include anti-cancer compounds, growth factors, differentiation factors, oligonucleotides, mRNA, or the like. Reagents include any compounds that facilitate the analysis of multicellular structures such as organoids. Exemplary reagents include, among others, labels, fixation agents, and clarifying agents. Labels may include dyes (e.g., visible stains and / or photoluminescent dyes). A photoluminescent dye is any substance that emits light in response to illumination with electromagnetic radiation, such as excitation light.

[0088] Each reservoir 105 may have an open top to facilitate the introduction and removal of fluids using a fluid transfer device (e.g., a pipette). A lid may be provided for placement on the culture vessel 102 to cover the open top of each reservoir 105 during culture in the incubator. The lid may have a flange that vertically overlaps the upper region of each reservoir and, optionally, is configured to limit lateral movement of the lid when covering the reservoir without creating an interference fit. In some embodiments, the lid may be a cap that forms a fluid-tight seal on top of one or more reservoirs 105.

[0089] Vessel 102 may contain at least one module 109 within one or more of the vessel's compartments. FIG. 1A shows module 109 located within culture chamber 103, with possible alternative locations for the same module shown in phantom lines. More specifically, each module 109 may be located within culture chamber 103, reservoir 105, or slot 107, among others. Thus, each module 109 may be located within or adjacent to culture chamber 103. Each module 109 may be contained within culture vessel 102 or may be present in a set of functionally distinct modules 109 located outside culture vessel 102 and configured to be selectively installed within culture vessel 102 by a user. Each module 109 may independently be an active electric / magnetic module (e.g., an electrode module, an actuator module, a sensor module, a pump module, and / or an illumination (light emitting) module, among others) or a passive module (e.g., a permeable interface module, a scaffold module, or a dummy module), as further described below.

[0090] Culture system 100 may also include an incubator 110 for containing vessel array 101 (see FIG. 1). The incubator may be temperature controlled to a suitable culture temperature for multicellular structures 104, such as at least 25° C., 30° C., or 35° C., among others (see also FIG. 1A). The humidity and / or atmosphere inside incubator 110 may also be controlled to encourage growth and development of multicellular structures 104.

[0091] Culture system 100 may further include a fluid transport system 111, a detection system 112, and a conveyor 113, each of which may be located inside or outside incubator 110. Fluid transport system 111 is configured to add and / or remove liquid from each culture vessel 102 of vessel array 101, such as into and / or out of its respective reservoir 105. The fluid transport system may therefore include one or more pipettes, fluid supplies, and / or waste containers. Detection system 112 is configured to collect data related to the multicellular structures contained by vessel array 101, such as by optical detection. For example, detection system 112 may include a light source 114 for illuminating at least a portion of the multicellular structures within each culture vessel 102 and an image sensor 115 for capturing images of at least a portion of the multicellular structures. Conveyor 113 may be configured to move components of culture system 100 relative to one another. For example, conveyor 113 may be configured to move container array 101 as a unit or only a portion thereof within culture system 100. For example, conveyor 113 may be configured to move container array 101 or its culture containers 102 in and / or out of incubator 110, to and / or from fluid transport system 111 and / or to and / or from detection system 112. Alternatively, or in addition, conveyor 113 may be configured to remove and replace one or more lids covering container array 101, if present, when reservoirs 105 and / or slots 107 are being accessed.

[0092] Control circuitry 116 of culture system 100 powers and / or operates any suitable devices of the culture system. For example, control circuitry 116 may control each of fluid transport system 111, detection system 112, and / or conveyor 113 via wired or wireless communication, which may be one-way or two-way communication. Each module 109 carried by vessel array 101 may also be wirelessly controlled by control circuitry 116, as indicated by the dashed arrow at 117. This wireless control is advantageous because it simplifies the construction of vessel array 101, allows the vessel array to be functionally customized as needed by installing appropriate modules, and eliminates the need for wires or electrical conductors that extend into the culture vessel and provide a pathway for microbial contamination.

[0093] 2 illustrates an exemplary vessel array 201 for the culture system 100 of FIG. 1. The vessel array comprises a plurality of vessel strips 218 (interchangeably referred to as vessel assemblies), each including a row of culture vessels 202 attached to one another, for example, via a common (shared) housing. A vessel strip may have any suitable number of culture vessels 202, such as at least two, three, four, or more. Each vessel strip 218 is received and held by a frame 219, optionally forming a row of vessel strips 218. Each vessel strip 218 may be placed as a unit into an individual receiving site of the frame 219, for example, during manufacturing or by a user. In the depicted embodiment, the frame 219 has a row of eight receiving sites for receiving the corresponding number of vessel strips 218, although in other embodiments, the frame may be configured to receive at least two, three, or more vessel strips 218 in the corresponding number of receiving sites. Each container strip 218 may be individually removable from the frame 219 to allow manipulation, processing, and / or analysis of the container strip (and / or the contents therein) separately from the other container strips 218 in the container array 201.

[0094] 3 shows another exemplary vessel array 301 for the culture system 100 of FIG. 1. The vessel array comprises a plurality of separate individual culture vessels 302 received and held by a frame 319. Each culture vessel 302 may be placed into a separate opening in the frame 319, for example, during manufacturing or by a user. The culture vessels may or may not be removable from the frame.

[0095] 4 illustrates the culture vessel 102 of FIG. 1A and a module set 420 of functionally distinct modules 409a-409c that may be contained within the culture vessel 102. Module set 420 may consist of any suitable number of functionally distinct modules, such as at least two, three, four, or more. Each module of module set 420 may have any suitable combination of properties, as described above with respect to module 109 or elsewhere herein (e.g., in Sections I, III, IV, and VI). Two or more modules of module set 420 may be interchangeably positionable within culture vessel 102 relative to one another, such as in the same compartment, as illustrated using phantom arrows for the placement of modules 409b and 409c in slot 107, and / or at least one module of module set 420 may be interchangeably positionable within each of two or more compartments of culture vessel 102, as illustrated using a pair of phantom arrows for module 409a within culture chamber 103 or reservoir 105.

[0096] 5 illustrates an exemplary culture vessel 502 and exemplary control circuitry 516 for the culture system 100 of FIG. 1. The culture vessel 502 is shown in a schematic side view and includes a culture chamber 503 containing multicellular structures 504, i.e., organoids 521, and an associated scaffold 522 for supporting organoid formation and / or growth. The scaffold 522 is attached to the bottom wall of the culture chamber 503 in FIG. 5, but in other embodiments, it may be attached to any suitable side or top wall of the culture chamber. At least one pair of reservoirs 505a, 505b is located across the culture chamber 503 and communicates with the culture chamber via individual channels 506a, 506b. Each reservoir is "vertically above" the culture chamber, which may mean that a vertical line extends through the reservoir and the culture chamber. Each reservoir 505a, 505b holds a respective culture medium 523a, 523b, which may have the same or different composition as each other. Culture chamber 503 also holds a culture medium, which may be supplied, at least in part, by one or both reservoirs 505a, 505b via one or both channels 506a, 506b.

[0097] The culture vessel 502 optionally defines a slot 507 located across the culture chamber 503. The slot 507 may be described as an access slot because the slot may communicate with the culture chamber 503 at its bottom end via an opening 508. The opening 508 may (or may not) have a diameter greater than that of each channel 506a, 506b, such as by at least 50% or 100%. The slot 507 is configured to receive a slot module 509a, which may be installed in the slot 507 of the culture vessel 502 during manufacturing or by a user. In some cases, a user may select a slot module 509a for installation in the slot 507 from a set of functionally distinct slot modules (see, e.g., FIG. 4 ). The ability to interchange slot modules of different functions allows the culture vessel 502 to be adapted for different culture / testing configurations for various types of organoids or other multicellular structures. In some cases, a dummy module may be installed in slot 507 to cover opening 508 when a slot module is not in use in the slot.

[0098] Slot 507 may have any suitable location relative to culture chamber 503 and reservoirs 505a, 505b. The slot may be centrally located between the reservoirs, as shown, or may have a lateral location relative to the reservoirs.

[0099] Slot 507 and reservoirs 505a, 505b may be open at their top ends. This configuration allows for placement of slot module 509a into slot 507 and dispensing of culture medium 523a, 523b into one or both reservoirs. Thus, vessel 502 may include a removable lid for covering the open tops of slot 507 and / or reservoirs 505a, 505b. Further aspects of lids for culture vessels and vessel assemblies are described below in Section V.

[0100] Culture chamber 503 may contain at least one chamber module 509b. The chamber module may be installed into a receptacle 524, such as via its top side, bottom side, or side, and the receptacle 524 may be converted into a culture chamber 503 by at least partially closing the top side, bottom side, or side of the receptacle. In some cases, if chamber module 509b is sufficiently large, the chamber module may be confined within culture chamber 503 unless and until the culture chamber is opened and / or disassembled by removing wall portions thereof.

[0101] The control circuitry 516 is configured to wirelessly control the slot module 509a and / or the chamber module 509b, as shown at 517, if one or both are electric / magnetic modules (see Section III). The control circuitry may include one or more antennas 525 to transmit / receive power and / or data to / from each module 509a, 509b, which is an electric module, if present, using near-field radiation. One or more coils 526 of the control circuitry may be utilized to generate a magnetic field to drive movement of at least the magnetic portion of each module 509a, 509b, if present. The control circuitry 516 may also comprise a computer, including a processor 527, one or more controllers 528, a memory storage device 529, and / or a user interface 530 (e.g., a display, keyboard, mouse, printer, and / or the like).

[0102] The control circuitry 516 may be configured to control the movement and / or operation of one or more electric / magnetic modules contained within each culture vessel of the array of culture vessels. Thus, the control circuitry may have at least one individual antenna 525 and / or at least one individual coil 526 for each culture vessel of the array.

[0103] 6 is a flowchart 630 of exemplary steps 631a-631j, which may be performed in any suitable order and combination to provide a method of culturing a multicellular structure. The method may be performed using any suitable systems, devices, cells, and scaffolds of the present disclosure.

[0104] A scaffold may be placed in a receptacle or culture chamber of the culture vessel in step 631 a. The scaffold may be placed by forming the scaffold in the receptacle or culture chamber, such as by 3D printing, or by placing a pre-formed scaffold in the receptacle or culture chamber. In some examples, the scaffold may be attached to a scaffold module, which may be placed in a slot in the culture vessel before or after the culture chamber is formed.

[0105] Cells may be introduced into the receptacle or culture chamber of the culture vessel in step 631b. The cells may include stem cells that are intended to generate an organized multicellular structure by differentiation, division, migration, etc. In other cases, the cells may be introduced into the receptacle or culture chamber as a preformed multicellular structure (e.g., an organism, tissue explant, tumor, organoid, or the like). The cells may be introduced during step 631a, or may be introduced before or after step 631a.

[0106] One or more modules may be installed in or adjacent to the receptacle or culture chamber in step 631c. Any combination of modules as disclosed herein may be installed. Any suitable number of one or more modules may be installed during manufacture of the vessel and / or by the user.

[0107] A culture chamber of the culture vessel may be formed in step 631d. The culture chamber may be formed by using a receptacle, which may have an open side, to at least partially close the open side. For example, the open side may be covered by joining a seal member to the receptacle on the open side (e.g., to the open bottom side of the receptacle) or by placing an insert into a shell that includes the receptacle. In either case, the chamber may be cooperatively formed using the receptacle and the seal member or insert.

[0108] A module may be placed in a slot of the container in step 631e. The module may be placed before or after step 631d. In some cases, steps 631a and 631e may be performed together using a scaffold module including a preformed scaffold. In some cases, steps 631b and 631e may be performed together using a module including cells.

[0109] Culture medium may be added to one or more reservoirs of the culture vessel in step 631f. Once added, the culture medium may optionally be gravity-driven to flow from one of the reservoirs into the culture chamber. Gravity may also drive the flow of culture medium out of the culture chamber and into different reservoirs of the culture vessel.

[0110] The vessel and its contents may be incubated in step 631g. Incubation may be carried out at a suitable temperature, in a suitable gaseous atmosphere, and for any suitable length of time, such as at least 1, 2, 3, 4, or 5 days, or at least 1, 2, or 3 weeks, among others.

[0111] If present, each electric / magnetic module in the culture vessel may be wirelessly controlled by the control circuitry in step 631h. This control may include transmitting power, force, and / or data to the module to drive movement of the module or at least a portion thereof and / or to control operation of the module. Steps 631g and 631h may be performed simultaneously.

[0112] Multicellular structures may be formed, grown, stimulated, and / or sensed within the culture chamber in step 631i, which may be performed in response to steps 631g and / or 631h.

[0113] A first module contained by the vessel may be removed and replaced with a second module in 631j. The first module may be removed from a slot in the vessel and replaced with a second module in the same slot. The first and second modules may be functionally different from each other. After replacing the first module with the second module, steps 631h and 631i may be repeated.

[0114] 7 is a flowchart 730 of exemplary steps 731ab, 731d, 731f, 731g, and 731i, which may be performed in any suitable order and combination, optionally adding one or more steps from flowchart 630 of FIG. 6 to provide a method of culturing multicellular structures. The method may be performed using any suitable systems, devices, cells, and scaffolds of the present disclosure. Steps in flowchart 730 that correspond to those in flowchart 630 have the same letter designation in both flowcharts.

[0115] The scaffold and / or cells may be disposed within the shell of the container in step 731ab. More specifically, the scaffold and / or cells may be disposed within a receptacle formed by a portion of the shell.

[0116] A container insert may be placed into the shell in step 731d to form a culture chamber. The culture chamber may be cooperatively formed by the receptacle and the shell. The receptacle may provide the bottom and side walls of the chamber, and the insert may provide the top wall of the culture chamber. Step 731d may be performed before or after the scaffold and / or cells are placed in the receptacle.

[0117] Culture medium may be added to at least one reservoir across (e.g., vertically above) the culture chamber in step 731f. The inserter may provide at least one reservoir across the culture chamber, or each reservoir.

[0118] The incubation vessel and its contents may be incubated in step 731g. Incubation may be carried out at a suitable temperature, in a suitable gaseous atmosphere, and for any suitable length of time, as described above with respect to step 631g (see FIG. 6).

[0119] 6 and 7 may be performed using an array of culture vessels. Thus, each step of the method may be performed on the culture vessels of the array as a group or on the culture vessels individually, as appropriate. III. Electrical / Magnetic Module

[0120] This section describes exemplary electric / magnetic modules for use in the culture systems and methods of the present disclosure (see Figures 8-17). Each electric / magnetic module is configured to be wirelessly powered / operated by a control circuit while the module is located within the culture vessel. The electric / magnetic modules utilize electricity, magnetism, or a combination thereof, wirelessly supplied or applied to the module by the control circuit, to drive movement of at least a portion of the module relative to the vessel and / or operation of at least one electric / electronic device of the module. The modules may be configured to be contained within any culture vessel of the present disclosure and within any compartment thereof, such as a slot, reservoir, and / or culture chamber of the culture vessel.

[0121] 8 illustrates an exemplary electrode module 809 (i.e., an electric / magnetic embodiment of module 109) and also illustrates a power antenna 825a and a communication antenna 825b of control circuitry 816. Antennas 825a, 825b are each configured to transmit power to electrode module 809 to operate it.

[0122] Electrode module 809 includes a housing 832 that supports electrode interface 833. The electrode interface may be located on any suitable side of housing 832, such as the bottom side as shown, or a lateral side thereof (e.g., if electrode module 809 is contained within a culture chamber), among others. Electrode interface 833 may include any suitable number of electrodes of any suitable shape. For example, electrode interface may have a pair of plate electrodes 834a that may be configured to contact the surface of the multicellular structure within the culture chamber and / or a pair of pin electrodes 834b that may be configured to penetrate and extend into the multicellular structure. Electrode interface 833 may be used to electrically stimulate the multicellular structure and / or to sense electrical properties of the multicellular structure from the outside or inside. Exemplary uses for the electrode module 809 include, among others, electrically stimulating (a) cardiomyocytes or neurons for pacing or activation, (b) myoblasts to promote muscle differentiation and growth in three dimensions, or (c) neural tissue (e.g., brain organoids) to promote axonal growth and morphological changes to affect network formation. Other exemplary uses for the electrode module 809 include sensing the electrical activity of any suitable cells / tissues, such as neurons, smooth muscle, cardiomyocytes, or skeletal muscle.

[0123] The housing 832 may contain any suitable electronic circuitry for wirelessly powering, communicating with, and / or controlling the electrode module 809. The circuitry may include a power receiver 835 having an antenna for receiving power wirelessly transmitted from a power antenna 825a of the control circuit 816. The circuitry may also include an internal communication antenna 836 for receiving signals from and / or transmitting signals to an external communication antenna 825b of the control circuit 816 to enable communication between the electrode module 809 and the control circuit 816. Any suitable communication protocol, such as at least one near-field communication (NFC) protocol, may be utilized. The electronic circuitry may also include a power storage unit 837 for storing power from the power receiver 835, a controller 838, and a digital-to-analog converter (DAC) and pulse generator 839. The electronic circuitry of any module in Section III may be sealed inside the housing to prevent damage if the module comes into contact with a liquid, such as a culture medium.

[0124] 9 illustrates an exemplary sensor module 909 (i.e., an electric / magnetic embodiment of module 109) and also illustrates a communication antenna 925 of control circuitry 916. Antenna 925 is configured to transmit power to sensor module 909 to operate it. Thus, a separate power antenna and power receiver may not be necessary (compare FIG. 8).

[0125] Sensor module 909 includes a housing 932 that supports a sensor interface 940. The sensor interface may be located on any suitable side of housing 932, such as the bottom side as shown, or a lateral side thereof (e.g., if sensor module 909 is contained within a culture chamber), among others. Sensor interface 940 may be configured to sense and measure any suitable physical or chemical parameter, such as temperature, movement, electrical parameters (e.g., potential, current, impedance, etc.), electric / magnetic fields (e.g., using Hall-effect sensors), pH, chemical potential, oxygen or carbon dioxide concentration, chemicals (e.g., using electrochemical sensors), etc. Sensor module 909 may be preferred when remote sensing is difficult or not possible, or when it is advantageous to establish direct contact between sensor interface 940 and the multicellular structure to be analyzed.

[0126] The housing 932 may contain any suitable electronic circuitry for enabling wireless powering, communication with, and / or control of the sensor module 909. The circuitry may include an internal communication antenna 936 for receiving signals from and / or transmitting signals to an external communication antenna 925 of the control circuitry 916 to power and operate the sensor module 909. Any suitable communication protocol may be utilized, such as at least one near-field communication (NFC) protocol. The electronic circuitry may also include a controller 938, an analog-to-digital converter (ADC) 941, a memory 942, and amplifier and sensor electronics 943.

[0127] 10 illustrates an exemplary pump module 1009 (i.e., an electric / magnetic embodiment of module 109) and also illustrates a power antenna 1025 of control circuitry 1016. Power antenna 1025 is configured to transmit power to pump module 1009. A separate communication antenna may or may not be present within control circuitry 1016 (compare FIG. 8).

[0128] The pump module 1009 includes a housing 1032 containing a pump 1044 for driving fluid flow. The pump 1044 is in fluid communication with a pair of nozzles 1045a, 1045b located on the periphery of the pump module 1009. Operation of the pump 1044 draws fluid into nozzle 1045a (acting as an inlet) and pushes fluid out of nozzle 1045b, or vice versa if the pump is driven in reverse. This movement of fluid may, among other things, enhance the flow of culture medium within the culture vessel and / or generate turbulence to stimulate organoid development. Use of the pump module 1009 may be preferred when gravity-driven flow is not applicable or is inefficient (e.g., due to the nature of the medium).

[0129] The housing 1032 may contain any suitable electronic circuitry for enabling it to wirelessly receive power from the control circuitry 1016 to operate the pump 1044. The circuitry may include a power receiver 1035 for receiving power transmitted wirelessly from the power antenna 1025, such as by inductive or capacitive coupling. The electronic circuitry may also include a power storage unit 1037 and a pump driver 1046.

[0130] 11 shows an exemplary culture system 1100 (see also FIG. 10 ) including a pair of pump modules 1009a, 1009b contained within a culture vessel 1102. The vessel 1102 includes a culture chamber 1103 containing organoids 1121. Located vertically above the culture chamber 1103 is a slot 1107 housing three compartments: a pair of reservoirs 1105a, 1105b holding separate culture media 1123a, 1123b, and a dummy module 1109 (an example of module 109 in FIG. 1 ). Reservoir 1105a communicates with the culture chamber 1103 via a pair of channels 1106a, 1106c, and reservoir 1105b communicates with the culture chamber 1103 via a pair of channels 1106b, 1106d.

[0131] Pumps 1009a and 1009b are driven by power transmitted from power antennas 1025a and 1025b, respectively, of control circuit 1016. Pump 1009a drives culture medium 1123a from reservoir 1105a through channel 1106c and into culture chamber 1103. Pump 1009b drives culture medium from culture chamber 1103 through channel 1106d and into reservoir 1105b. Thus, there is a net pump-driven flow of medium from reservoir 1105a to reservoir 1105b. The pumps can be driven in reverse to move medium from reservoir 1105b back to reservoir 1105a, or this may be done via gravity-driven flow through channels 1106a and 1106b.

[0132] 12 shows a culture system 1200 for mechanically stimulating organoids 1221 or other multicellular structures. The culture system 1200 includes a container 1102 (see FIG. 11), a control circuit 1216, and a pair of magnetic modules 1209a, 1209b contained within the culture chamber 1103 of the container 1102. The organoids 1221 may be located between the magnetic modules. The magnetic modules can function as magnetic actuators for applying alternating mechanical strain to the organoids, and the driving principle is a magnetic force applied by the control circuit 1216 to create a linear drive.

[0133] The electrode module 809 may be located in the slot 1107, with both pin electrodes 834b extending into the organoid 1221 (see also FIG. 8). However, as discussed below, mechanical stimulation of the organoid 1221 provided by movement of the magnetic modules 1209a, 1209b driven by the control circuit 1216 does not require the presence of the electrode module 809.

[0134] Each magnetic module 1209a, 1209b includes a permanent magnet 1247, which may be encapsulated by a housing 1232. The housing may have a surface coating that promotes attachment to the organoids 1221. The magnets 1247 of magnetic modules 1209a, 1209b each have a north (N) and a south (S) pole, with a magnetic axis extending through both poles. The magnetic modules 1209a, 1209b may be arranged within the cell chamber 1103 so that the magnetic axes of the magnetic modules are coaxial with each other, anti-parallel as shown, creating magnetic repulsion, or parallel, creating magnetic attraction.

[0135] The control circuit 1216 includes at least one coil (the counterpart of the fixed coil 1226a, 1226b) that can be electrically energized to create one or more additional magnetic fields. The additional magnetic fields effectively strengthen or reduce the attraction or repulsion between the magnetic modules 1209a, 1209b, thereby driving the movement of the magnetic modules toward or away from each other. Each coil 1226a, 1226b defines a coil axis that can be oriented parallel to the magnetic axis of the magnetic modules 1209a, 1209b. Figure 13 illustrates the energization of both coils 1226a, 1226b, indicated by current arrows 1348, and the generation of the magnetic field polarity shown in the coils. Magnetic repulsion between coil 1226a and magnetic module 1209a, and between coil 1226b and magnetic module 1209b, drives movement of the magnetic modules toward each other, as indicated by the movement arrows at 1349, which applies compression to the organoid 1221. In other cases, energizing coils 1226a, 1226b may drive magnetic modules 1209a, 1209b farther apart, which may apply tension to the organoid if attached to both modules. In still other cases, the magnetic modules may be used for magnetic sensing of organoid movement. The ability to apply repeated mechanical stress through the magnetic modules is particularly beneficial for certain types of organoids, such as bone and muscle organoids, which may require this stress for proper development.

[0136] 14 and 15 schematically illustrate how the magnetic drive mechanism of FIGS. 12 and 13 can be implemented in a culture system 1400 including an array of culture vessels 1402a-1402c. (The locations of the culture vessels are indicated generally with dashed arrows, but the vessels themselves are omitted for ease of illustration.) Each culture vessel 1402a-1402c contains an individual organoid 1421a-1421c sandwiched between a pair of magnetic modules 1209a, 1209b, as described above for the single culture vessel 1102. Control circuitry 1416 provides a series of electrically energizable coils 1426a-1426d aligned along the same lines as the vessels 1402a-1402c, organoids 1421a-1421c, and each pair of magnetic modules 1209a, 1209b. The coil axes defined by the coils 1426a-1426d extend along the rows of the containers 1402a-1402c, respectively.

[0137] The positions of the magnetic modules 1209a, 1209b are shown in FIG. 14 without and in FIG. 15 with the electrical energization of the coils 1426a-1426d. The electrical energization is indicated using the arrows at 1448 in FIG. 15. As explained above with respect to the single culture vessel of FIGS. 12 and 13, the appropriate electrical energization of the coils 1426a-1426d drives each pair of magnetic modules 1209a, 1209b linearly toward each other along the line of the vessels 1402a-1402c, allowing the organoids 1421a-1421c to be mechanically stimulated synchronously.

[0138] 16 is a schematic top view of a culture system 1600 having a different configuration of electrically energizable coils 1626 for driving the movement of the magnetic modules 1209a, 1209b of FIGS. 14 and 15. (Only a subset of the coils 1626 are identified using numerical identifiers.) The culture system 1600 includes an array of vessels 1602a-1602c. (The vessels' locations are generally indicated using dashed arrows, but the vessels themselves are omitted for ease of illustration.) Each vessel 1602a-1602c contains an individual organoid 1602a-1602c sandwiched between a pair of magnetic modules 1209a, 1209b, as described above with respect to the culture system 1400. However, the magnetic axis of each pair of magnetic modules 1209a, 1209b is oriented orthogonally, e.g., horizontally, relative to the array of vessels 1602a-1602c, as shown. The control circuit 1616 provides at least one row or pair of rows of coils 1626. Each coil 1626 may define a coil axis that is parallel to the magnetic axis of the magnetic module. Appropriate electrical energization of the coils 1626 drives the magnetic modules 1209a, 1209b linearly in a direction parallel to the rows of vessels 1602a-1602c, mechanically stimulating the organoids 1621a-1621c.

[0139] 17 shows an exemplary optical module 1709 (i.e., an electric / magnetic embodiment of module 109) configured to be wirelessly powered and controlled by control circuitry 1716 via its power antenna 1725. Optical module 1709 includes a housing 1732 containing a power receiver 1735 for receiving power from power antenna 1725, a power storage unit 1737, and a light source 1750. The light source generates optical radiation that may be used for optical stimulation (e.g., via optogenetics) of multicellular structures within the enclosure and / or illumination for optical detection of the multicellular structures.

[0140] Exemplary light sources include light emitting diodes, lasers, or the like. The light source may also include any suitable optics for directing or focusing light. For example, the light source may have a waveguide, such as an optical fiber, to direct light from the light module 1709 onto the surface of the multicellular structure and / or to extend into the multicellular structure to illuminate the multicellular structure from the inside.

[0141] The optical module 1709 may be used to facilitate imaging of the multicellular structures contained within the culture chamber of the culture vessel. For example, the optical module may provide brightfield or darkfield illumination of the multicellular structures when appropriately positioned within the culture vessel, such as within its reservoir, slot, or culture chamber. IV. Passive Modules

[0142] This section describes exemplary passive modules for use in the culture systems and methods of the present disclosure (see Figures 18-20). The passive modules are configured to function without a source of electrical power and without interaction with control circuitry. Each passive module may be configured to be installed in a specific compartment of the vessel, or alternatively, in each of two or more compartments of the vessel.

[0143] FIG. 18 is a somewhat schematic diagram of an exemplary scaffold module 1809, which is an embodiment of module 109 of FIG. 1A. The scaffold module includes a body 1851 and a scaffold 1822 attached to the body 1851. The scaffold 1822 is configured to support the formation and growth of multicellular structures, such as organoids. The scaffold may be formed on the body 1851, such as by 3D printing, or may be formed first and then attached to the body 1851. In either case, the scaffold 1822 may be mounted on any suitable side of the body 1851, such as the bottom, top, or lateral side, among others. The scaffold module 1809 may be located within a slot in the culture vessel, such as by being placed into the slot during manufacture of the culture vessel or by a user. In some cases, the scaffold module may be an inserter that forms one or more reservoirs in the culture vessel, as further described below in Section V. Scaffolding modules may be used to mount and support scaffolding and to keep the scaffolding in place, allowing for improved handling and scaffolding addition and removal.

[0144] FIG. 19 shows an exemplary permeable interface module 1909, which is one embodiment of module 109 of FIG. 1A. The permeable interface module is cross-sectionally shown in FIG. 19 to reveal its internal structure. The permeable interface module 1909 has a hollow body 1951 defining a cavity 1952 that is continuous with an inlet 1953 and an interface opening 1954. The inlet 1953 may be plugged with a flexible cap 1955. A permeable member 1956, such as a permeable membrane or gel, is attached to the hollow body 1951 at the interface opening 1954 to create a permeable interface 1957 (e.g., a permeable wall) on any suitable side of the body 1951, such as the bottom, top, or side, among others. The permeable interface 1957 optionally and selectively allows passage of fluids and / or small molecules into and out of the cavity 1952 via the permeable interface.

[0145] The permeable interface module 1909 may contain any suitable culture medium within the cavity 1952. The culture medium may be a gas, liquid, gel, or the like. The culture medium may have a different phase, composition, and / or chemical potential than the culture medium present in the culture chamber of the container. The permeable interface 1957 may be positioned in physical contact with the multicellular structures and / or culture medium within the container. As an example, the permeable interface module 1909 may contain air (or other gas) within the cavity 1952, creating a gas / liquid interface at the permeable member 1956 and enabling the culture of lung organoids within the culture chamber of the culture container. In other examples, the permeable interface module 1909 may contain any suitable chemical that can be discharged through the permeable member 1956 into the culture chamber of the culture container.

[0146] 20 shows a permeable interface module 1909 operatively positioned within the slot 1107 (see also FIG. 11 ) of the container 1102, adjacent to the opening 1108 and over the lung organoids 2021. The cavity 1952 is filled with gas such that the permeable member 1956 forms a gas-liquid interface between the gas in the cavity 1952 and the liquid culture medium in the culture chamber 1103.

[0147] A passive module contained within a compartment of a culture vessel may be described as a dummy module. A dummy module may be a substitute. Like any of the modules of the present disclosure, a dummy module may function to reduce the fluid holding capacity of the vessel and / or block or seal a slot across the culture chamber. However, a dummy module may also provide a surface shape (concave or convex), surface chemistry / texture (e.g., hydrophilic, microstructured, etc.), and / or functional surface that is advantageous for the culture protocol. V. Vessel Assembly

[0148] This section describes exemplary vessel assemblies, vessel arrays formed with the vessel assemblies, and culture vessels of the vessel assemblies for use in the culture systems and methods of the present disclosure (see Figures 21-40).

[0149] 21 shows an exemplary container array 2101 for culturing a corresponding array of multicellular structures, such as organoids. The container array 2101 comprises at least one container strip 2118 held by a frame 2119. While only one container strip 2118 is shown in FIG. 21 , the frame 2119 is configured to removably hold two, three, or more container strips 2118, which may be substantially identical to one another. In the depicted embodiment, the frame 2119 defines eight receptor sites 2158 for a corresponding number of container strips 2118 arranged in a row, although only a subset of the receptor sites 2158 may be occupied by a container strip 2118 at any given time. Frame 2119 may have a footprint corresponding to that of a standard microplate (a standard microplate footprint is 127.71 mm x 85.43 mm), with the length and width of the frame's footprint being within 10% and 5%, respectively, of that of a standard microplate. This correspondence between frame 2119 and the standard microplate footprint achieves mechanical compatibility with incubators, analytical instrumentation, and handling systems for standard microplates.

[0150] The vessel strip 2118 forms a row of at least two, three, or more culture vessels, such as four culture vessels 2102a-2102d in the depicted embodiment. Each culture vessel 2102a-2102d includes an individual culture chamber 2103 formed by a lower region of the culture vessel.

[0151] 22 shows vessel strip 2118 being removed from frame 2119 to allow imaging of the multicellular structures contained within culture chamber 2103. Removal of vessel strip 2118 from frame 2119 allows the vessel strip to be properly positioned relative to imaging system 2112 (or other detection system) without interference from frame 2119 or other vessel strips 2118 held by the frame.

[0152] Each culture chamber 2103 has one or more optical windows formed by one or more walls of the culture chamber. Each optical window is configured to be transparent to optical radiation, such as visible light, and may have smooth, optionally planar, inner and outer surfaces to minimize scattering of the optical radiation. In the depicted embodiment, each culture chamber 2013 has a bottom optical window 2159a and a pair of side optical windows 2159b, 2159c arranged opposite each other across the culture chamber 2103 (see also FIGS. 24, 26, and 30).

[0153] FIG. 22 illustrates how imaging system 2112 may capture images of multicellular structures contained within culture vessel 2102c. Thin sections of the multicellular structures may be illuminated through side optical window 2159b (and / or window 2159c) of the culture vessel using light source 2114, which may generate light sheet 2160. Light (e.g., fluorescence) from the multicellular structures may be collected using objective lens 2161, which collects light propagated through bottom optical window 2159a of culture vessel 2102c. Thus, imaging system 2112 may perform selective plane illumination microscopy (SPIM), also known as light sheet microscopy. In other examples, illumination may be performed through bottom optical window 2159a and light collection from one of side optical windows 2159b or 2159c. In still other examples, illumination may be performed using an optical module located within the culture vessel (see, e.g., Section III). In still other embodiments, imaging system 2112 may utilize two-photon excitation microscopy, tomography, or the like.

[0154] Reservoir strip 2118 provides individual pairs of reservoirs 2105a, 2105b and individual slots 2107 located across the culture chamber 2013 of each vessel 2102a-2102d (see FIGS. 21 and 22). Slot modules 2109 may be located within slots 2107 and may be any of the electric / magnetic or passive modules disclosed herein (see, e.g., Sections I, III, and IV).

[0155] 23 shows an exploded view of the container strip 2118. The container strip comprises a shell 2162 (interchangeably referred to as a housing), a plurality of inserters 2163 configured to be received within the shell 2162, and a lid 2164 configured to cover the open top side of the shell 2162 and / or the inserters 2163. In some embodiments, the lid 2164 may not be required. A separate pair of magnetic modules 1209a, 1209b may be located within the shell 2162 below each inserter 2163 (see also Section III), although any other suitable module may be located within the shell below the inserter, or no module may be located within the shell below the inserter.

[0156] Shell 2162 may be internally divided into multiple sections, such as sections 2165a-2165d. Sections 2165a-2165d may be integrally formed with one another. Sections 2165a-2165d may be directly attached to one another or may be separated from one another using individual spacer regions 2166 located intermediate each adjacent pair of sections.

[0157] Each section 2165a-2165d of shell 2162 includes a lower region forming a receptacle 2167 and an upper region forming a receiving space 2168 for one of the inserts 2163 (see FIGS. 23, 26, and 28-30). Each culture chamber 2103 is cooperatively formed using shell 2162 and one of the inserts 2163, which may capture a module (in the depicted embodiment, a counterpart of magnetic modules 1209a, 1209b (see FIG. 26)) within the culture chamber. Receptacle 2167 provides a side wall of culture chamber 2103, and one of the inserts 2163 provides an upper wall portion of the culture chamber. A scaffold for the organoids may be disposed within the receptacle 2167 of each inserter 2163, such as by being formed or installed therein (e.g., attached to the bottom or side wall of the receptacle) before the inserter 2163 is assembled with the shell 2162. In other cases, the scaffold may be disposed on the bottom side of the inserter 2163, such as by being 3D printed thereon or attached after scaffold formation before the inserter is assembled with the shell 2162. In still other cases, the scaffold may be provided by module 2109 (see also Section IV).

[0158] Each inserter 2163 is configured to fit into one of the sections 2165a-2165d. Thus, the outer dimensions of the inserter may correspond to the dimensions of the receiving space 2168. The receptacle 2167 may have one or more horizontal dimensions smaller than the receiving space 2168 and form a shoulder 2169 at the bottom of the receiving space 2168 to support the inserter 2163 and prevent further advancement downward into the section (see FIGS. 26, 29, and 30).

[0159] Each inserter 2163 may be locked within the shell 2162 by a snap-fit ​​mechanism that engages when the inserter is seated within the shell (see FIGS. 27 and 31-33). The inserters may define protrusions 2170 that are received within respective openings 2171 defined by the side walls of the shell 2162, or the inserters may define openings and the shell may define protrusions.

[0160] Each insert 2163 defines various openings at its bottom. The openings include at least one channel, a counterpart of channels 2106a, 2106b, at the bottom of each reservoir 2105a, 2105b (see FIG. 33). Each channel 2106a, 2106b extending from reservoir 2105a or 2105b provides fluid communication between one of the reservoirs and the culture chamber 2103 located vertically below the reservoir (see also FIG. 26). The insert also defines openings 2108 at the bottom ends of slots 2107, providing fluid communication between the slots 2107 and the culture chamber 2103 (see FIGS. 26, 33, 34).

[0161] FIG. 35 shows a lid assembly 2172 for a container array 2101 (see also FIG. 21). The lid assembly 2172 includes a series of lids 2164 for covering a corresponding number of container strips 2118 held by a frame 2119 (see also FIG. 26). Each lid 2164 may fit over the top of a container strip (e.g., the top of a shell 2162) and may have a peripheral flange that overhangs downward and is configured to vertically overlap and horizontally surround the container strip at its upper edge. Maintaining sterility can be a major issue during the entire incubation and testing process, which can take up to several months. Therefore, it would be advantageous for the lid assembly 2172 to block the entry of contaminating microorganisms through the open tops of the reservoirs 2105 a, 2105 b and slots 2107 of each container 2102 a-2102 d of each container strip 2118. The lid assembly 2172 allows all of the container strips to be covered by the same lid assembly, although individual lids 2164 may be removed from the lid assembly 2172. For example, the lid assembly 2172 may include a carrier 2173 to which each lid 2164 is removably attached (e.g., via a weak adhesive, an interference fit, or a snap-fit ​​mechanism, among others). This configuration allows for easy removal of any single container strip 2118, including its lid 2164, from the other container strips 2118 and their lids 2164 of the container array 2101, such as for imaging or other processing. In other embodiments, the carrier 2173 may be omitted, and the lids 2164 may be configured to be integrally formed with one another and removable by breaking the frangible connections joining adjacent pairs of lids 2164 to one another.

[0162] 36 and 37 show another exemplary inserter 3663 for installation into the shell 2162 (see also FIG. 23). The inserter 3663 is the same as the inserter 2163, except that the inserter 3663 includes a gasket 3674 configured to create a fluid-tight seal with the shell 2162. The gasket 3674 may be formed from a softer, more deformable material (e.g., an elastomer) and may be attached to a body 3675 formed from a harder, less deformable material. In some cases, the gasket 3674 may be fabricated on the body 3675 by overmolding. The gasket 3674 may be located on a lateral side of the body 3675, such as around a lower region of the body 3675, or on a bottom side of the body 3675, among others.

[0163] Figure 38 shows yet another exemplary insert 3863 for installation into shell 2162 (see also Figure 23). Insert 3863 forms a pair of reservoirs 3805a, 3805b, which are adjacent and share side walls with each other instead of being separated by a central slot (compare Figure 33). Insert 3863 can be advantageous if a scaffold will be attached to the bottom side of the insert because there is more surface area available for attachment. Insert 3863 also allows the channel at the bottom of the insert to be more centrally located for vertical alignment with a multicellular structure centered in the culture chamber below the insert.

[0164] Figure 39 shows yet another exemplary insert 3963 (see also Figure 23) for placement into shell 2162. Insert 3963 forms four reservoirs 3905a-3905d and a central slot 3907 located midway between pairs of reservoirs (compare Figure 33). Insert 3963 can be advantageous when the multicellular structure is being fed internally and externally with different culture media.

[0165] Figure 40 shows yet another exemplary inserter 4063 for installation into shell 2162 (see also Figure 23). Like inserter 3963, inserter 4063 forms four reservoirs 4005a-4005d, but lacks the central slot located midway between pairs of reservoirs (compare Figure 39). Inserter 4063 has more than two reservoirs and combines the potential advantages of centrally locating a channel throughout a multicellular structure. VI. Selected Aspects

[0166] This section describes selected aspects of the systems, methods, and devices of the present disclosure as a series of indexed paragraphs.

[0167] Paragraph A1. A system for culturing multicellular structures such as organoids, comprising: (a) a container including a culture chamber for containing the multicellular structures (such as organoids); (b) an electric / magnetic module configured to be coupled to and / or located within the container, optionally at a location within or adjacent to the culture chamber; and (c) control circuitry configured to wirelessly power and / or operate the electric / magnetic module, optionally the electric / magnetic module being removably coupled to and / or removably located within the container, and optionally the electric / magnetic module being capable of being received within each of two or more compartments of the container, optionally the two or more compartments being selected from the culture chamber, one or more reservoirs, and / or slots.

[0168] Paragraph A2. The system of paragraph A1, wherein the control circuitry is configured to wirelessly power and / or operate the electric / magnetic module using near-field radiation.

[0169] Paragraph A3. The system of paragraph A2, wherein the control circuit is configured to wirelessly transfer power to the electric / magnetic module via inductive coupling or capacitive coupling.

[0170] Paragraph A4. The system of paragraph A2 or A3, wherein the control circuit is configured to communicate wirelessly with the electric / magnetic module via at least one short-range wireless communication protocol.

[0171] Paragraph A5. A system described in any of paragraphs A1-A4, wherein the electric / magnetic module is contained within or configured to be contained within the culture chamber and includes a magnet, and the control circuit is configured to create a magnetic field that drives movement of the magnet within the culture chamber.

[0172] Paragraph A6. The system described in paragraph A5, wherein the electric / magnetic module is a first module, the magnet is a first magnet, and further comprising a second module including a second magnet, wherein the first and second modules are contained or configured to be contained within the culture chamber simultaneously, and the control circuit is configured to drive movement of the first and second magnets relative to each other, optionally towards and / or away from each other within the culture chamber.

[0173] Paragraph A7. The system of any of paragraphs A1-A6, wherein the electric / magnetic module includes a sensor.

[0174] Paragraph A8. The system of paragraph A7, wherein the electric / magnetic module includes a chemical sensor, an electrical sensor, an optical sensor, and / or a temperature sensor.

[0175] Paragraph A9. The system of any of paragraphs A1-A8, wherein the electric / magnetic module includes an electrode.

[0176] Paragraph A10. The system of paragraph A9, wherein the electrodes are configured to electrically stimulate and / or electrically sense the multicellular structure within the culture chamber.

[0177] Paragraph A11. The system of any of paragraphs A1-A10, wherein the electric / magnetic module includes a light source.

[0178] Paragraph A12. The system of paragraph A11, wherein the light source is configured to illuminate at least a portion of the multicellular structure within the culture chamber.

[0179] Paragraph A13. The system of any of paragraphs A1-A12, wherein the electric / magnetic module includes a pump configured to drive fluid flow into and / or out of the culture chamber.

[0180] Paragraph A14. A system described in any of paragraphs A1-A13, wherein the electric / magnetic module is located or configured to be located within a slot adjacent to the culture chamber, and optionally the container defines an opening that provides fluid communication between the slot and the culture chamber.

[0181] Paragraph A15. The system of paragraph A14, wherein the container includes two or more reservoirs in fluid communication with the culture chamber, optionally a slot located vertically above the culture chamber, optionally between at least a pair of the two or more reservoirs, optionally each of the two or more reservoirs in separate communication with the culture chamber, optionally each of the two or more reservoirs is not connected to any external source of liquid, such as via tubing, optionally each of the two or more reservoirs and the culture chamber are formed by and / or located within the same housing, and optionally each of the two or more reservoirs is directly attached to the culture chamber and / or shares a wall with the culture chamber.

[0182] Paragraph A16. A system described in paragraph A14 or A15, wherein the electric / magnetic module is included in a set of two or more modules that perform different functions from each other and are configured to be coupled to and / or positioned within the container, and optionally interchangeably positionable within the same compartment of the container, such as the same reservoir, culture chamber, and / or slot.

[0183] Paragraph A17. Optionally, the containers each include at least one or two or more reservoirs located across (optionally vertically above) the culture chamber and optionally each sharing a wall with the culture chamber, optionally the two or more reservoirs being formed integrally with each other and / or by the same insert, optionally the electric / magnetic module being located vertically midway between the upper side of the two or more reservoirs and the bottom of the culture chamber, and / or the system further optionally comprises a removable lid configured to be placed on the container to cover at least one or two or more reservoirs.

[0184] Paragraph A18. The system of any of paragraphs A1-A17, further comprising a container assembly interconnected to form a container array containing containers, each container in the container array comprising an individual culture chamber for containing a multicellular structure (such as an organoid), optionally the containers in the container array being substantially identical to one another, and optionally the electric / magnetic module being movable between the containers in the container array.

[0185] Paragraph A19. The system of paragraph A18, wherein the container assembly has a length corresponding to the length or width of a standard microplate footprint.

[0186] Paragraph A20. The system of paragraph A18 or A19, further comprising a container array including a container assembly and forming at least two or three rows of containers, each container of the at least two or three rows of containers including an individual culture chamber for containing a multicellular structure.

[0187] Paragraph A21. The system of any of paragraphs A18-A20, further comprising a frame for holding a plurality of container assemblies, the frame optionally having a length and / or width corresponding to the length and / or width of a standard microplate footprint, and optionally the plurality of container assemblies being substantially identical to one another, and optionally the plurality of container assemblies including a corresponding plurality of shells that are substantially identical to one another.

[0188] Paragraph A22. The system of any of paragraphs A1-A21, wherein the vessel includes an optical window formed by a wall of the culture chamber.

[0189] Paragraph A23. The system of any of paragraphs A1-A22, further comprising a scaffold positioned or configured to be positioned within the culture chamber and configured to support organoid formation within the culture chamber.

[0190] Paragraph A24. The system of any of paragraphs A1-A23, wherein the container is provided by a device of any of paragraphs C1-C20.

[0191] Paragraph B1. A method of culturing multicellular structures such as organoids, comprising: (a) containing the multicellular structures within a culture chamber of a container, wherein an electric / magnetic module is optionally removably coupled to and / or located within the container at a location within or adjacent to the culture chamber; and (b) wirelessly powering / operating the electric / magnetic module using control circuitry, wherein optionally the electric / magnetic module is removably coupled to and / or located within the container, and optionally the electric / magnetic module is removably coupled to and / or located within two or more of the container. can be received in each of the more than two compartments, optionally wherein two or more compartments are selected from a culture chamber, one or more reservoirs, and / or slots, and optionally wherein the multicellular structures are organoids at least 0.2, 0.5, 1, or 2 mm in diameter and are formed / grown within the culture chamber without connecting the container to an outside source of fluid (e.g., culture medium) (e.g., via tubing and / or tubes) and / or without electrically connecting the container to a wire or other electrical conductor.

[0192] Paragraph B2. The method of paragraph B1, wherein the step of powering / operating includes a step of wirelessly transmitting electrical energy to the electrical / magnetic module.

[0193] Paragraph B3. The method of paragraphs B1 or B2, wherein the powering / operating step is performed at least in part via inductive or capacitive coupling of the control circuit and the electric / magnetic module to each other.

[0194] Paragraph B4. The method of any of paragraphs B1-B3, wherein the powering / operating step includes a step of wirelessly communicating with the electric / magnetic module using near-field radiation.

[0195] Paragraph B5. The method of paragraph B4, wherein the step of wirelessly communicating includes exchanging data with the electric / magnetic module using at least one short-range wireless communication protocol.

[0196] Paragraph B6. A method as described in any of paragraphs B1-B5, wherein the electric / magnetic module includes a magnet and the step of powering / operating includes a step of driving movement of the magnet within the culture chamber using a magnetic field created by a control circuit.

[0197] Paragraph B7. The method of paragraph B6, wherein the culture chamber contains a pair of electric / magnetic modules, each including a magnet, and wherein the step of powering / operating includes a step of driving the electric / magnetic modules relative to each other, optionally towards and / or away from each other within the culture chamber.

[0198] Paragraph B8. A method described in any of paragraphs B1-B7, wherein the powering / operating step includes using a sensor of the electric / magnetic module to sense properties of the multicellular structure (e.g., located within the culture chamber) and / or culture medium in contact with the multicellular structure.

[0199] Paragraph B9. The method of any of paragraphs B1-B8, wherein the step of powering / operating includes a step of electrically stimulating the multicellular structure.

[0200] Paragraph B10. The method of any of paragraphs B1-B9, wherein the powering / operating step includes driving fluid flow into and / or out of the culture chamber.

[0201] Paragraph B11. The method of any of paragraphs B1-B10, wherein the electric / magnetic module is located within the culture chamber.

[0202] Paragraph B12. The method of any of paragraphs B1-B11, wherein the electric / magnetic module is located at least primarily (ie, more than half by volume) within the vessel and outside the culture chamber.

[0203] Paragraph B13. The method of paragraph B12, wherein the electric / magnetic module is located in a slot defined by the container.

[0204] Paragraph B14. The method of any of paragraphs B1-B13, further comprising collecting data associated with the multicellular structure while the multicellular structure remains within the culture chamber.

[0205] Paragraph B15. The method of paragraph B14, wherein collecting data includes capturing an image of at least a portion of the multicellular structure.

[0206] Paragraph B16. The method of paragraphs B14 or B15, wherein the step of collecting data is performed using a sensor of the module.

[0207] Paragraph B17. The method of any of paragraphs B1-B16, further comprising disposing an electric / magnetic module within a compartment of the vessel.

[0208] Paragraph B18. The method of paragraph B17, wherein the step of placing includes capturing the electric / magnetic module within the culture chamber.

[0209] Paragraph B19. The method of paragraph B17, wherein the disposing step includes placing an electric / magnetic module within the vessel but outside the culture chamber, optionally within a reservoir or slot of the vessel.

[0210] Paragraph B20. The method of any of paragraphs B17-B19, optionally further comprising a step of selecting an electric / magnetic module from a set of two or more functionally distinct modules located outside the container prior to the placing step based on (i) the type of organoids present in and / or to be cultured in the culture chamber, (ii) the culture protocol or protocol stage selected for the organoids, (iii) test conditions for the organoids, and / or (iv) parameters to be sensed for the organoids and / or culture medium in contact with the organoids.

[0211] Paragraph B21. The method of paragraph B20, further comprising selecting two or more functionally distinct modules from the set, wherein the placing step comprises, for each selected module, coupling the selected module to a container and / or placing the selected module within the container.

[0212] Paragraph B22. The method of paragraph B20 or B21, wherein the set of two or more functionally distinct modules includes at least one passive module, and optionally, the selecting step includes selecting a passive module.

[0213] Paragraph B23. The method of any of Paragraphs B1-B22, wherein the method is carried out using a system of any of Paragraphs A1-A24.

[0214] Paragraph C1. A device for culturing multicellular structures such as organoids, comprising: (a) a shell, optionally having an open top; and (b) an inserter including at least one reservoir or two or more reservoirs, the inserter being configured to be received within the shell, optionally via the open top, such that the shell and the inserter cooperatively form a culture chamber for the multicellular structure, the culture chamber being located below (optionally vertically below) the two or more reservoirs and optionally in fluid communication with each of the at least one reservoir or two or more reservoirs via individual channels defined by the inserter.

[0215] Paragraph C2. The device of paragraph C1, further comprising a lid configured to be placed on the shell to cover the at least one reservoir and / or each reservoir of the two or more reservoirs.

[0216] Paragraph C3. The device of paragraph C2, wherein the lid is configured to completely cover the open top of the shell.

[0217] Paragraph C4. The device of either paragraph C1 or C3, further comprising a scaffold configured to support organoid formation within the culture chamber.

[0218] Paragraph C5. The device of paragraph C4, wherein the scaffolding is attached to a wall of the shell.

[0219] Paragraph C6. The device of paragraph C4, wherein the shell and inserter each further comprise a module that is part of the container and provides a scaffold, the module configured to be (optionally removably) coupled to and / or positioned within a compartment of the container, such as a slot defined by the inserter.

[0220] Paragraph C7. The device of paragraph C6, wherein the slot is located vertically above the culture chamber and between at least a pair of reservoirs of the two or more reservoirs.

[0221] Paragraph C8. A device described in any of paragraphs C1-C7, wherein the shell includes a section having an upper region defining a receiving space and a lower region forming a receptacle, the inserter being configured to be received within the receiving space, and the receptacle being configured to cooperatively form a culture chamber with the inserter.

[0222] Paragraph C9. The device of paragraph C8, wherein the inserter is configured to form a fluid-tight seal with a plurality of side walls and / or a bottom wall of the upper region of the compartment.

[0223] Paragraph C10. The device of paragraph C9, wherein the inserter includes a body and a gasket attached to the body, the gasket configured to engage each side wall and / or bottom wall of the plurality of side walls and form a fluid-tight seal.

[0224] Paragraph C11. The device of paragraph C10, wherein the gasket is molded onto the body.

[0225] Paragraph C12. The device of any of paragraphs C1-C11, wherein the inserter forms a slot and defines an opening at a bottom end of the slot, and further comprises a module positioned or configured to be positioned within the slot vertically above the opening, and optionally the module is an electric / magnetic module.

[0226] Paragraph C13. The device of any of paragraphs C1-C12, wherein the device comprises a plurality of inserters, and the shell forms an array of sections, each configured to receive an individual inserter of the plurality of inserters, and each section and individual insert cooperatively form a culture chamber for the multicellular structure.

[0227] Paragraph C14. The device of paragraph C13, in which the segments of the array of segments are integrally formed with one another as a single piece.

[0228] Paragraph C15. The device of paragraph C13 or C14, further comprising a lid configured to be placed on the shell to cover the array of segments.

[0229] Paragraph C16. The device of any of paragraphs C1-C15, wherein a lower region of the shell forms at least one optical window of the culture chamber.

[0230] Paragraph C17. The device of paragraph C16, wherein a lower region of the shell forms at least two optical windows of the culture chamber.

[0231] Paragraph C18. The device of paragraph C17, wherein the at least two optical windows include a bottom window and at least one side window.

[0232] Paragraph C19. The device of paragraph C17 or C18, wherein the at least two optical windows comprise a pair of side windows arranged opposite each other.

[0233] Paragraph C20. A device described in any of paragraphs C1-C19, wherein the shell and inserter each form part of the same container and each further comprise one or more modules, optionally a set of two or more functionally distinct modules, such as a set including at least one or at least two electric / magnetic modules and / or at least one or at least two passive modules, configured to be coupled to and / or positioned within the container, and optionally at least two of the functionally distinct modules configured to be interchangeably installed in the same compartment of the container (e.g., the same reservoir, culture chamber, or slot).

[0234] Paragraph D1. A method for culturing a multicellular structure such as an organoid, the method comprising: (a) placing an insert, optionally including at least one reservoir or two or more reservoirs, into a shell and using the insert and shell to cooperatively form a culture chamber, wherein the culture chamber is optionally located below (e.g., vertically below) and in fluid communication with each of the at least one reservoir or two or more reservoirs; and (b) culturing the multicellular structure in the culture chamber.

[0235] Paragraph D2. The method of paragraph D1, further comprising placing a lid on the shell to cover the two or more reservoirs.

[0236] Paragraph D3. The method of paragraph D1 or D2, wherein the culture chamber is in fluid communication with each reservoir of the two or more reservoirs through a channel defined by the insert.

[0237] Paragraph D4. The method of any of Paragraphs D1-D3, further comprising placing a scaffold within the shell, the scaffold configured to promote organoid formation within the culture chamber, and optionally, cells of the multicellular structure embedded within the scaffold.

[0238] Paragraph D5. The method of Paragraph D4, wherein the placing step optionally includes forming a scaffold within the shell before placing the inserter.

[0239] Paragraph D6. The method of Paragraph D4, wherein the placing step includes placing a preformed scaffold within the shell.

[0240] Paragraph D7. The method of Paragraph D6, wherein the step of placing the preformed scaffold within the shell is performed before placing the inserter.

[0241] Paragraph D8. The method of paragraph D6, wherein the step of installing the preformed scaffold in the shell is performed by or after installing the inserter, and optionally, the preformed scaffold is already attached to the bottom side of the inserter before installing the inserter.

[0242] Paragraph D9. The method of any of paragraphs D1-D8, wherein the shell includes an array of compartments, the placing step includes placing at least two inserters within the array of compartments to form two or more separate culture chambers, and the culturing the multicellular structure includes culturing individual organoids in each of the two or more separate culture chambers.

[0243] Paragraph D10. The method of any of Paragraphs D1-D9, further comprising collecting data associated with the multicellular structure while the multicellular structure remains within the culture chamber.

[0244] Paragraph D11. The method of any of Paragraphs D1-D10, wherein collecting data includes capturing an image of at least a portion of the multicellular structure.

[0245] Paragraph D12. The method of any of Paragraphs D1-D11, wherein the method is performed using any of the devices described in Paragraphs C1-C20.

[0246] Paragraph E1. A system for culturing multicellular structures such as organoids, comprising: (a) a container including a culture chamber for containing the multicellular structures and at least one reservoir or at least two or more reservoirs and optional slots, each in fluid communication with the culture chamber; and (b) two or more modules having different functions from each other and configured to be interchangeably coupled to and / or disposed within the container.

[0247] Paragraph E2. The system of paragraph E1, wherein the container defines an opening that communicates with the culture chamber at a bottom end of the slot.

[0248] Paragraph E3. The system of paragraph E2, wherein each module of the two or more modules is configured to be positioned adjacent to the opening when installed in the slot.

[0249] Paragraph E4. The system of any of paragraphs E1-E3, wherein the two or more modules include a first module having a permeable membrane.

[0250] Paragraph E5. The system of Paragraph E4, wherein the membrane is configured to form an interface between the liquid in the culture chamber and the liquid or gas in the first module.

[0251] Paragraph E6. The system of any of paragraphs E1-E5, wherein at least one of the two or more modules is an electric / magnetic module.

[0252] Paragraph E7. The system of Paragraph E6, wherein at least one module includes a module having an electronic device.

[0253] Paragraph E8. The system of paragraph E6 or E7, wherein at least one of the two or more modules does not include an electronic device.

[0254] Paragraph E9. The system of any of paragraphs E1-E8, wherein the container is provided by a device of any of paragraphs C1-C20.

[0255] Paragraph F1. A method for culturing a multicellular structure such as an organoid, the method comprising the steps of: (a) containing the multicellular structure within a culture chamber of a container, the container defining a slot communicating with the culture chamber, a first module positioned within the slot; (b) removing the first module from the slot; and (c) installing a second module within the slot, the second module configured to perform a different function than the first module.

[0256] Paragraph F2. The method of Paragraph F1, wherein the container defines an opening that provides communication between the slot and the culture chamber.

[0257] Paragraph F3. The method of paragraphs F1 or F2, wherein at least one of the first and second modules extends from the slot into the culture chamber through an opening.

[0258] Paragraph F4. The method of any of Paragraphs F1-F3, wherein at least one of the first and second modules includes an electronic device.

[0259] Paragraph F5. The method of any of Paragraphs F1-F4, wherein the first and second modules include a membrane that forms an interface between the liquid in the culture chamber and the liquid or gas in the module.

[0260] Paragraph F6. The method of any of Paragraphs F1-F5, wherein at least one of the first and second modules includes an electrode that contacts the multicellular structure and / or liquid in the culture chamber.

[0261] Paragraph F7. The method of any of paragraphs F1-F6, further comprising using a control circuit to wirelessly power / operate one of the first and second modules while the one module is positioned in the slot.

[0262] Paragraph F8. The method of any of paragraphs F1-F7, wherein the container is provided by a device of any of paragraphs C1-C20.

[0263] Paragraph G1. An organoid culture vessel that does not require the attachment of tubes and wires to the vessel itself.

[0264] Paragraph H1. A method of organoid culture, comprising forming / growing organoids (e.g., large organoids) in a container without connecting the container to an external source of fluid (e.g., via one or more tubes, such as tubing) and / or without electrically connecting the container to an external electrical conductor (e.g., an electrical conductor electrically connected to an electric / magnetic device and / or control circuit).

[0265] While the present invention has been described through the above examples and features, it will be understood by those skilled in the art that numerous modifications, combinations, and variations of the examples and features can be made without departing from the inventive concepts disclosed herein. Furthermore, the present invention should not be considered limited to any specific objectives or embodiments described herein, but rather should be considered applicable to accomplish a wide variety of objectives other than those described herein. This disclosure has described several examples of the present technology with reference to the accompanying drawings, in which only some of the possible examples are shown. However, other aspects may be embodied in many different forms and should not be construed as limited to the examples described herein, even if not specifically exemplified in combination. Rather, these examples are provided so that this disclosure will be thorough and complete, and will fully convey the scope of possible examples to those skilled in the art.

Claims

1. 1. A system for culturing multicellular structures, comprising: a container comprising a culture chamber configured to contain the multicellular structure; an electric / magnetic module configured to be positioned within the vessel; a control circuit configured to wirelessly power and / or operate the electric / magnetic module; a container assembly forming an array of containers, the array of containers being interconnected and comprising the containers, each container of the array of containers comprising a separate culture chamber for containing a multicellular structure; a frame for holding a plurality of container assemblies including the container assembly, the frame having a length and / or width corresponding to the length and / or width of a standard microplate footprint; wherein the electric / magnetic module is contained within or configured to be contained within the culture chamber and includes a magnet, and the control circuitry is configured to create a magnetic field that drives movement of the magnet within the culture chamber.

2. The system described in claim 1, wherein the containers in the row of containers are substantially identical to each other.

3. The system of any one of claims 1 to 2, wherein the control circuitry is configured to wirelessly transfer power to the electric / magnetic module via inductive or capacitive coupling.

4. The system of claim 1 , wherein the electrical / magnetic module includes a chemical sensor, an electrical sensor, an optical sensor, and / or a temperature sensor.

5. The system of any of claims 1 to 4, wherein the electric / magnetic module includes a pump configured to drive fluid flow into and / or out of the culture chamber.

6. A system described in any of claims 1 to 5, further comprising an additional electric / magnetic module, the additional electric / magnetic module being located or configured to be located within a slot adjacent to the culture chamber.

7. The system described in claim 6, wherein the container defines an opening that provides fluid communication between the slot and the culture chamber.

8. The system of any one of claims 6 to 7, wherein the container includes two or more reservoirs in fluid communication with the culture chamber, and the slot is located vertically above the culture chamber.

9. The system described in claim 8, wherein the slot is located between at least a pair of the two or more reservoirs.

10. A system described in any one of claims 8 to 9, wherein each of the two or more reservoirs is separately connected to the culture chamber.

11. 1. A method for culturing a multicellular structure, comprising: containing multicellular structures within a culture chamber of a vessel, wherein an electric / magnetic module is located within the vessel at a location within the culture chamber, the vessel being contained within a vessel assembly, the vessel assembly forming an array of interconnected vessels; wirelessly powering / operating said electric / magnetic module using a control circuit; using a frame to hold a plurality of container assemblies including said container assembly, said frame having a length and / or width corresponding to the length and / or width of a standard microplate footprint; wherein the electric / magnetic module includes a magnet, and powering / operating includes using a magnetic field created by the control circuit to drive movement of the magnet within the culture chamber.

12. The method of claim 11, wherein the containers in the row of containers are substantially identical to each other.

13. The method of any one of claims 11 to 12, wherein powering / operating comprises wirelessly transmitting electrical energy to the electric / magnetic module.

14. 14. The method of any one of claims 11 to 13, wherein powering / operating is performed at least in part via inductive or capacitive coupling of the control circuit and the electric / magnetic module to each other.

15. 15. The method of any of claims 11-14, wherein powering / operating comprises sensing properties of the multicellular structure and / or culture medium within the culture chamber using a sensor of the electric / magnetic module.

16. The method of any of claims 11 to 15, wherein powering / operating comprises driving fluid flow into and / or out of the culture chamber.

17. The method of any of claims 11-16, further comprising collecting data relating to the multicellular structure while the multicellular structure remains within the culture chamber.

18. The method of claim 17 , wherein collecting data is performed using sensors on the module.

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