Systems, methods, and devices for culturing multicellular structures

Abstract

To provide systems, methods and devices for culturing a multicellular structure such as an organoid.SOLUTION: An exemplary system comprises a vessel, an electric / magnetic module and a control circuit. The vessel may include a culture chamber to contain a multicellular structure. The electric / magnetic module may be configured to be located in the vessel, at a position in or adjacent the culture chamber. The control circuit may be configured to wirelessly power and / or operate the electric / magnetic module.SELECTED DRAWING: None

Description

TECHNICAL FIELD

[0001] The present application claims priority from U.S. Provisional Application No. 62 / 976,151, filed on February 13, 2020, the entire content of which is incorporated herein by reference. BACKGROUND ART

[0002] Organoids ("mini-organs") are three-dimensional aggregates of different cell types generated in vitro that share a certain degree of similarity with organs, including exhibiting realistic histology of organ-specific tissues. Cell aggregates can be generated by seeding a small number of stem cells in a matrix. Stem cells then proliferate, differentiate and self-organize within the matrix while using the matrix as a scaffold. With this approach, organoids resembling tissues from brain, heart, intestine, kidney, liver and stomach, among others, have been generated to date. These promising results suggest that organoid culture provides new insights into organ development and function, and has the potential to replicate disease models that enable in vitro drug screening. Organoids can revolutionize the way drugs are discovered and therapies are personalized.

[0003] Despite the growing importance of organoids, efficient culture of them remains a challenge. New systems, methods and devices for culturing organoids and other multicellular structures are needed. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0004] This disclosure provides systems, methods, and devices for culturing multicellular structures such as organoids. An exemplary system comprises a container, an electrical / magnetic module, and a control circuit. The container may include a culture chamber for containing the multicellular structure. The electrical / magnetic module may be configured to be located within the container, within the culture chamber, or adjacent thereto. The control circuit may be configured to wirelessly power and / or operate the electrical / magnetic module. The present invention provides, for example, the following items: (Item 1) A system for culturing multicellular structures, A container including a culture chamber configured to contain the multicellular structure, An electrical / magnetic module configured to be located within the container, either inside or adjacent to the culture chamber, A control circuit configured to wirelessly power and / or operate the aforementioned electrical / magnetic module A system that includes these features. (Item 2) The system according to item 1, wherein the control circuit is configured to wirelessly transmit power to the electrical / magnetic module via inductive coupling or capacitive coupling. (Item 3) The system according to any one of items 1-2, wherein the electrical / magnetic module is contained within or configured to be contained within the culture chamber, includes a magnet, and the control circuit is configured to create a magnetic field that drives the movement of the magnet within the culture chamber. (Item 4) The electrical / magnetic module is the system described in item 1, including a chemical sensor, an electrical sensor, an optical sensor, and / or a temperature sensor. (Item 5) The system according to any one of items 1-4, wherein the electrical / magnetic module includes a pump configured to drive fluid flow in and / or out of the culture chamber. (Item 6) The electrical / magnetic module is located in or configured to be located in 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, according to any one of items 1-5. (Item 7) The system according to item 6, wherein the container comprises two or more reservoirs that are in fluid communication with the culture chamber, the slots optionally located vertically above the culture chamber, between at least one pair of the two or more reservoirs, and optionally each of the two or more reservoirs is in separate communication with the culture chamber. (Item 8) The system according to any one of items 1-7, further comprising a container assembly that is interconnected and forms a row of containers containing the containers, wherein each container in the row of containers contains an individual culture chamber for containing a multicellular structure, and optionally, the containers in the row of containers are substantially identical to each other. (Item 9) The system according to any one of items 6-8, further comprising a frame for holding a plurality of container assemblies, including the container assembly, wherein the frame optionally has a length and / or width corresponding to the length and / or width of a standard microplate footprint. (Item 10) A method for culturing multicellular structures, The container contains a multicellular structure within its culture chamber, and the electrical / magnetic module is located within the container, either within or adjacent to the culture chamber. Using a control circuit, the electrical / magnetic module is wirelessly powered / operated. Methods that include... (Item 11) The method according to item 10, wherein supplying / operating power includes wirelessly transmitting electrical energy to the electrical / magnetic module. (Item 12) The method according to item 10 or 11, wherein power supply / operation is carried out at least in part via inductive or capacitive coupling between the control circuit and the electrical / magnetic module. (Item 13) The method according to any one of items 10-12, wherein the electrical / magnetic module includes a magnet, and powering / operating it involves using a magnetic field created by the control circuit to drive the movement of the magnet within the culture chamber. (Item 14) The method according to any one of items 10-13, wherein power supply / operation includes using sensors of the electrical / magnetic module to sense the properties of the multicellular structure and / or culture medium within the culture chamber. (Item 15) The method according to any of items 10-14, wherein power supply / operation includes driving fluid flow in and / or out of the culture chamber. (Item 16) The method according to any one of items 10-15, further comprising collecting data related to the multicellular structure while the multicellular structure remains in the culture chamber. (Item 17) The method of item 16, wherein data collection is performed using the sensors of the module. (Item 18) A device for culturing multicellular structures such as organoids, wherein the device is A shell with an open top, An inserter comprising two or more reservoirs, wherein the inserter is configured to be received into the shell via an open top so that the shell and the inserter cooperate to 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 separate channels defined by the inserter. A device equipped with the following features. (Item 19) The device according to item 18, further comprising a scaffold configured to support organoid formation within the culture chamber. (Item 20) The device according to item 19, provided by a module, wherein the scaffold is located in or configured to be located within a slot defined by the inserter. (Item 21) The device according to any one of items 18-20, wherein the shell comprises a division having an upper region defining a receptive space and a lower region forming a receptacle, the inserter is configured to be received within the receptive space, and the receptacle is configured to cooperate with the inserter to form the culture chamber. (Item 22) The inserter comprises a module that forms a slot, defines an opening at the bottom end of the slot, and is configured to be located or positioned vertically above the opening within the slot, and optionally the module is an electrical / magnetic module, as described in any of items 18-21. (Item 23) A system for culturing multicellular structures, wherein the system is A container comprising a culture chamber for containing the multicellular structure, and two or more reservoirs and slots, each having fluid communication with the culture chamber, Two or more modules having different functions from each other and configured to be interchangeably installed in the slots, and A system that includes these features. (Item 24) The system according to item 23, wherein the container defines an opening at the bottom end of the slot that communicates with the culture chamber. (Item 25) The two or more modules mentioned above include a first module having a permeable membrane, as described in any of items 23-24. [Brief explanation of the drawing]

[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 comprising a container array for containing multicellular structures in individual culture vessels, at least one electrical / magnetic module positioned in at least one culture vessel of the container array, and a control circuit for powering and / or operating the electrical / magnetic module via wireless transmission between the electrical / magnetic module and the control circuit.

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

[0007] [Figure 2] FIG. 2 is an exploded schematic top view of an embodiment of the container array of FIG. 1.

[0008] [Figure 3] FIG. 3 is an exploded schematic top view of another embodiment of the container array of FIG. 1.

[0009] [Figure 4] FIG. 4 is a schematic view of the culture vessel of FIG. 1A and a set of functionally different modules for use in the culture vessel.

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

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

[0012] [Figure 7] Figure 7 is a flowchart of exemplary steps that may be performed in a method for culturing a multicellular structure using a culture vessel, which includes a shell and an inserter.

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

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

[0015] [Figure 10] Figure 10 is a somewhat schematic diagram of an exemplary pump module for use in the culture system of Figure 1 or the method of Figure 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 schematic diagram of a selected side of a partially cross-sectional view of the implementation of the system in Figure 1, including two copies of the pump module in Figure 10 contained within the culture chamber of the culture vessel, adjacent to the organoid.

[0017] [Figure 12] Figure 12 is a partially schematic diagram of a selected side of a partially cross-sectional view of an implementation of the system of Figure 1, which includes a control circuit, the culture vessel of Figure 11, and a pair of magnetic modules contained within the culture chamber of the culture vessel, with the organoid positioned between the magnetic modules.

[0018] [Figure 13]Figure 13 is another diagram of the system implementation of Figure 12, obtained with the control circuit coils energized to drive the movement of the magnetic modules toward each other in order to mechanically stimulate the organoid.

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

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

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

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

[0023] [Figure 18] Figure 18 is a somewhat schematic diagram of an exemplary scaffolding module, including scaffolding attached to the main body of the module and configured to support the formation and growth of organoids.

[0024] [Figure 19] Figure 19 is a cross-sectional view of an exemplary permeable interface module for use in the culture system of Figure 1 or the method of Figure 6.

[0025] [Figure 20]Figure 20 is a cross-sectional view of the permeable interface module of Figure 19, which is operationally positioned within a slot in 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 a container assembly that forms a row of culture vessels, each containing a pair of magnetic modules, and each adjacent to a separate slot containing another module structurally and functionally different from the magnetic modules, the column containing a corresponding row of culture vessels including a magnetic chamber.

[0027] [Figure 22] Figure 22 is an isometric view of the culture device in Figure 21 after removal from the frame and operational matching of one of the culture vessels in the container assembly with the light source and objective lens of the imaging system.

[0028] [Figure 23] Figure 23 is an isometric view of the culture device of Figure 21, including a lid not shown in Figure 21 or Figure 22.

[0029] [Figure 24] Figure 24 is a side view of the culture device shown in Figure 21, obtained after separation.

[0030] [Figure 25] Figure 25 is a top view of the culture device shown in Figure 21, obtained after separation.

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

[0032] [Figure 27] Figure 27 is a side view of the culture device shell from Figure 21, obtained after separation.

[0033] [Figure 28] Figure 28 is a top view of the shell shown in Figure 27.

[0034] [Figure 29] Figure 29 is a cross-sectional view of the shell in Figure 28, generally obtained along line 29-29 in Figure 28 through one of the four divisions of the shell.

[0035] [Figure 30] Figure 30 is another cross-sectional view of the shell in Figure 28, generally obtained along line 30-30 in Figure 28.

[0036] [Figure 31] Figure 31 is a side view of the inserter of the culture device shown in Figure 21, obtained after separation.

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

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

[0039] [Figure 34] Figure 34 is a cross-sectional view of the inserter of Figure 31, generally obtained along line 34-34 of Figure 33.

[0040] [Figure 35] Figure 35 is an exploded view of a lid assembly for a container array, including a row of container assemblies held by the frame of Figure 21, each lid assembly containing a series of lids that are substantially the same as the lids of Figure 23.

[0041] [Figure 36] Figure 36 is a modified form of the inserter in Figure 31, including a gasket for creating a liquid-tight seal with the shell in Figure 27.

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

[0043] [Figure 38] Figure 38 is a top view of an exemplary slotless inserter for placement inside the shell of Figure 27 to collaboratively form a culture chamber with the shell.

[0044] [Figure 39] Figure 39 is a top view of an exemplary 4-reservoir inserter for placement inside the shell of Figure 27 to collaboratively form a culture chamber with the shell.

[0045] [Figure 40] Figure 40 is a top view of an exemplary slotless 4 reservoir inserter for placement inside the shell of Figure 27 to collaboratively form a culture chamber with the shell. [Modes for carrying out the invention]

[0046] Detailed explanation 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 cultivation, organoids go through different stages of the culture protocol as they grow, differentiate, and develop. Currently, most of these stages require manual interaction for feeding, monitoring, and handling. Since the culture vessels containing the organoids may not be suitable for monitoring the state of the developing organoids, equipment changes are often necessary. Culture protocols that rely on manual interaction are expensive and prone to process errors. Furthermore, culturing large organoids to develop specific interacting cell types may require not only treating the cells with appropriate compounds but also exposing the organoids to a suitable physical environment specific to the type of organoid being cultured. For example, cardiomyocytes and nerve cells 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 apparatuses that rely on tubing and wiring. However, these apparatuses are bulky, not standardized, and not easily scaled up to culture many organoids simultaneously.

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

[0048] This disclosure provides a system and method for culturing multicellular structures such as organoids. The system may comprise a container, including a culture chamber for containing the multicellular structure. An electrical / magnetic module may be configured to be located within the container, either within or adjacent to the culture chamber. A control circuit may be configured to power and / or operate the electrical / magnetic module by wireless transmission. In this method, the multicellular structure may be contained within the culture chamber of the container. The electrical / magnetic module may be located within the container, either within or adjacent to the culture chamber. The electrical / magnetic module may be powered / operated using a control circuit via wireless transmission of power, force, and / or signals (e.g., data). The electrical / magnetic module may include magnets that can be 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 structure contained within the culture chamber. In other embodiments, the electrical / magnetic module may include electrodes for stimulating the multicellular structure, sensors for sensing the 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 equivalent.

[0049] The ability to wirelessly power / operate the electrical / magnetic module in this system and method is significant because it eliminates the need for wires or other electrical conductors extending from the control circuit to the container. As a result, the container becomes more portable with fewer constraints regarding where it is positioned relative to the control circuit and is more easily separated from the control circuit (e.g., for performing imaging procedures). Furthermore, this wireless approach can improve the fidelity and stability of experiments involving multicellular structures because it allows the electrical / magnetic module to be positioned very close to, or in contact with, the multicellular structure being analyzed. This system and method can also facilitate the automation of complex cultures of multicellular structures by providing the necessary physical environment and enable multi-channel in-situ monitoring of multicellular structures. This system and method may be capable of simultaneously stimulating, maintaining, and monitoring a large number of organoids in an automated manner.

[0050] The containers of this 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 in 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 enables, among other things, the cultivation of specific types of organoids, the implementation of specific culture protocols or protocol stages, the creation of desired test conditions, and / or onboard (in-container) 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 suit the specific needs of various users. Furthermore, the introduction of modules into the container allows the modules to be very close to, or in contact with, the multicellular structure, which generates a more direct interaction between the module and the multicellular structure. Moreover, the introduction of modules into the container may not result in any increase in the size of the occupied area. Therefore, the modified container using the module can remain compact, which allows more copies of the container to fit within the occupied area of ​​a standard microplate, and consequently, allows more multicellular structures to be cultured simultaneously in the incubator.

[0051] A device and method for culturing multicellular structures such as organoids are provided. The device may comprise a shell having an open top. The device may also comprise an inserter containing two or more reservoirs. The inserter may be configured to be received into the shell via the open top so that the shell and the inserter cooperate to form a culture chamber for the multicellular structure. The culture chamber may be located beneath the two or more reservoirs and may be in fluid communication with each of the two or more reservoirs via separate channels defined by the inserter. In this method, an inserter containing two or more reservoirs may be placed inside the shell, and the inserter and shell may cooperate to form a culture chamber. The culture chamber may be located beneath each of the two or more reservoirs and may be in fluid communication with them. The multicellular structure may be cultured within the culture chamber.

[0052] The devices and methods described in the preceding paragraphs may offer various advantages for culturing multicellular structures such as organoids, including any combination of the following: The inserters may be selected from a set of inserters having different characteristics from one another, such as different reservoir, channel, and / or slot configurations. Thus, the same shell may be assembled with different types of inserters, and the resulting container structure may be customized for the specific needs of the user. The inserters may also have reservoirs (and optional slots) positioned vertically above the culture chamber so that gravity can drive fluid flow in and out of the culture chamber. Furthermore, each shell may define a row of compartments capable of receiving an inserter, forming individual culture chambers. Thus, the shells and two or more inserters 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 an occupied area 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 in order to be processed separately from other container assemblies in the array.

[0053] The container may provide multiple reservoirs that are in fluid communication with the chamber via channels formed within a shared wall between the reservoir and the chamber. This configuration may be described as a standard feeding interface. In some embodiments, 3D printing provides a connection to any suitable printed structure of the standard feeding interface inside the container, enabling the growth of different types of organoids.

[0054] The matrix can provide a temporary scaffold for the appropriate type of cell as they develop into organoids. The cells may self-organize and generate their own extracellular matrix, which may replace some or all of the scaffold. The same may be true with respect to internal feeding, where the container may provide a general interface, which may optionally be modified by 3D printing, and the cells may self-organize to best utilize this modified interface.

[0055] In some embodiments, the scaffold (with or without cells) may be placed within the receptacle of the container body, and the culture chamber may optionally be formed from the receptacle using a sealing member while the container is inverted. Once these processes are complete, the container may be rotated with its right side up (relative to its organoid culture orientation), and at least one reservoir above the culture chamber may be filled with a supplying fluid. If no cells are yet present inside the scaffold, suitable cells may be placed in the supplying fluid and introduced into the scaffold together with the supplying fluid from the reservoir above the culture chamber.

[0056] The organoids to be formed may require an initial culture period before a specific feeding protocol can be initiated. The feeding protocol may involve filling the reservoir with a suitable medium and removing the medium from the reservoir according to a predetermined schedule and / or based on the organoid development stage or conditions. The feeding protocol may depend on the shape of the scaffold and the type of organoids to be formed.

[0057] The container may enable optical sheet 3D imaging. The culture chamber of the container may have two, three, or more optical windows, through which light may propagate in and / or out of the culture chamber. 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 pairs of side optical windows arranged opposite each other.

[0058] This disclosure enables the development of large functional organoids. Large organoids may exceed, among other things, about 0.1, 0.2, 0.5, 1, or 2 millimeters in average or maximum diameter. Working with large organoids remains challenging, and researchers face two main limitations. Firstly, each type of organoid may require different culture conditions, such as a concrete hydrogel as a scaffold, or even mechanical stimulation such as shear forces from culture medium flow. Secondly, microscopic examination of large organoids can be extremely difficult. State-of-the-art methods still involve sectioning, staining, and image acquisition of fixed samples using a confocal scanning microscope or even a slide reader for organoid material.

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

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

[0061] The technical terms used in this disclosure have meanings generally recognized by those skilled 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, cell lines (e.g., cell lines), primary cells, cells from tissue samples, transfected cells, cells from clinical samples (e.g., blood samples, fluid aspirates, tissue explants, etc.), cells that form an entire organism, and / or equivalents.

[0063] Any suitable cells may be introduced into the culture chamber (or into the receptacle which will form part of the culture chamber). The introduced cells may include stem cells (e.g., pluripotent stem cells), supporting cells, and / or equivalents. The cells may be deposited into the culture chamber or receptacle, and / or into a scaffold located within 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 equivalents.

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

[0065] Culture chamber - A compartment containing a multicellular structure and having walls on substantially all sides of a space that is almost or completely enclosed. At least one of the walls may define one or more openings to allow communication with and / or passage into 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, among other things, contain a carbon source (e.g., glucose), inorganic salts, vitamins, and growth regulators. The term “culture medium” as used herein means at least one culture medium, which may, for example, be a separate volume of culture medium, a first and second culture medium of different composition, a culture medium of substantially identical composition in contact with different / separate cell cultures, or a combination / mixture of previously separated volumes of the same culture medium.

[0067] Culture vessels—devices for culturing multicellular structures. A culture vessel (synonymously referred to as a vessel) may include a culture chamber and one or more reservoirs that are in fluid communication with the culture chamber. A vessel assembly or vessel array is a collection of culture vessels for culturing 1, 2, or 3-dimensional arrangements of multicellular structures. The culture vessels disclosed herein may be single-use devices (consumables) or reusable.

[0068] Exemplary – to serve as an illustration or example. Similarly, the term “to illustrate” means to illustrate by providing an example. Neither term implies desirability or superiority.

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

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

[0071] A module is a structurally and functionally discrete unit configured to be contained within a culture vessel. A module may be insertable into the culture vessel only while the culture vessel is intact or when the culture vessel is disassembled, and may be removable from or inremovable from the culture vessel. A module may be an active module, also called an electromagnetic module, which utilizes electricity and / or magnetism for operation and is optionally powered / operated by wireless transmission between a control circuit and the module. Alternatively, a module may be a passive module, which does not utilize either electricity or magnetism to perform its intended purpose. Electromagnetic modules include magnetic modules that have permanent magnets but no electrical / electronic devices, electrical modules that have electrical / electronic devices but no permanent magnets, and modules that have permanent magnets and electrical / electronic devices. A magnetic module may optionally require an externally generated, time-varying magnetic field for operation (e.g., to drive the movement of the magnetic module and / or its magnets).

[0072] Modules may have any preferred shape and size. Modules, particularly their housing or body, may be, for example, cubic (e.g., cube), cylindrical, conical, or equivalent. The shape of the module in cross-section may correspond to that of a slot or other compartment in the container, so that the module fits into that 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 to have the same standard size and shape for all modules regardless of the compartment's destination. 4×4×4mm 3 These are exemplary sizes of active and passive modules, corresponding to the sizes of larger organoids that may occur within the culture chamber of the container.

[0073] A multicellular structure is a three-dimensional arrangement of interconnected biological cells. A multicellular structure may also be an organized multicellular structure, consisting of different cell types arranged non-randomly relative to one another. Exemplary multicellular structures include organoids, organisms (at any developmental stage), tissue explants, tumors, or equivalents.

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

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

[0076] Organoids are three-dimensional aggregates of different types of cells that are generated outside of a living organism and exhibit some degree of similarity to organs, such as having a realistic histology of organ-specific tissues. 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 having an open side, optionally such as an open top, open bottom, or open side. The receptacle may be converted into a culture chamber by covering or closing at least partially the sides of the receptacle.

[0078] Scaffolds – extracellular support frameworks for the culture of multicellular structures. A scaffold is typically a matrix in which cells of a multicellular structure are embedded or will be embedded. The scaffold may be provided by one or more hydrogels. Each hydrogel may contain one or more thermoplastic structural components, such as, among others, Matrigel, alginate, nanofibrilized cellulose, collagen, fibrin, and / or polyethylene glycol, which cooperatively form the matrix in a temperature-dependent manner.

[0079] In some embodiments, two or more different hydrogels / matrices may be placed in the culture chamber of the container. The hydrogels / matrices may differ with respect to any preferred parameters such as melting temperature, resistance to enzymatic degradation, solubility, cell attraction and / or cell repulsion properties, and / or equivalents.

[0080] Each hydrogel / matrix may contain any preferred components. 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., aggrecan, agrin, brevican, collagen type XVIII, leprecan, neurocan, perlecan, small leucine-rich proteoglycan, versican, or GAGs bound to a core protein (e.g., via its serine) to form equivalents), fibrous proteins (e.g., collagen, elastin, fibronectin, laminin, etc.), and / or equivalents. Protease recognition sites (e.g., relating to scaffold metalloproteinases (MMPs)) may be incorporated into the hydrogel / matrix to enable cellular degradation / remodeling. The frequency of such sites, along with the sequence of each site, may be selected to enable a suitable amount of degradation / remodeling.

[0081] One or more growth factors may be present in the matrix when it is formed, or introduced into the culture medium after the matrix has formed. Exemplary growth factors that may be preferred include angiopoietin, bone morphogenetic protein (BMP), ciliary neurotrophic factor, colony-stimulating factor, ephrin, epidermal growth factor, erythropoietin, fibroblast growth factor, glial neurotrophic factor, hepatocyte growth factor, insulin, insulin-like growth factor, interleukin, leukemia suppressor factor, keratinocyte growth factor, neuregulin, neutrophin, platelet-derived growth factor, transforming growth factor, tumor necrosis factor (alpha), vascular endothelial growth factor, and / or equivalents. II. Overview of Culture Systems and Methods

[0082] This section provides an overview of the culture system and method of this disclosure (see Figures 1-7).

[0083] Figure 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 container array 101, which includes a set of culture vessels 102, of which only three are clearly identified in Figure 1. Each culture vessel 102 is configured to contain an individual multicellular structure. The container array 102 may be or include a linear array, a two-dimensional array (e.g., a rectangular array, a hexagonal array, etc., as shown), and / or a three-dimensional array. Each of the container arrays 101 and / or two or more discrete container assemblies may have an area occupied by the length and / or width of a standard microplate to facilitate mechanical and fluid handling using a robotic system designed for manipulating a standard microplate. The number of culture vessels 102 in the vessel array 101 is at least three in the first dimension and may be one or more in the second orthogonal dimension (for example, at least two, three, or more).

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

[0085] Each compartment of the culture vessel 102 may have any preferred 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, in particular, a multicellular structure such as an organoid of any preferred size, such as a multicellular structure having a diameter of at least 0.2, 0.5, 1, or 2 mm. In exemplary embodiments, each reservoir 105 of the culture vessel 102 has a volume of at least 2, 5, or 10 times the volume of the culture chamber, and / or a volume greater than that of the culture chamber 103, such as at least 0.5, 1, 2, 4, or 6 mL. 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 the same height as each reservoir 105. Each compartment of the vessel may be rectangular, circular, elliptical, or equivalent in horizontal cross-section. The rectangular cross-section of each compartment of the container may be advantageous because this shape allows for very efficient use of available space and enables the 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 preferred procedure. In exemplary embodiments, the culture vessel may consist of at least one polymer, which may include a transparent polymer. The culture vessel 102 may be formed from at least pairs of discrete components, such as a shell and an inserter or a body and a sealing member, which are formed integrally as a single piece, or which are fitted together to form a culture chamber 103, and / or are attached to each other. Thus, the culture chamber 103, reservoir 105, and / or slot 107 may have fixed positions relative to each other, and / or may be irremovably / rigidly attached to each other within the culture vessel 102.

[0087] The reservoir 105 of container 102 may hold any suitable substance to be supplied to the culture chamber 103. Exemplary substances include, among other things, nutrients, effectors, and reagents. Suitable nutrients include any substance to promote the health and proliferation of cells inside the culture chamber 103, and therefore 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, etc. Effectors include any molecule (such as inducers or inhibitors) that activates, controls, or inactivates a process or action (such as differentiation, protein synthesis, migration, etc.). Exemplary effectors include anti-cancer compounds, growth factors, differentiation factors, oligonucleotides, mRNA, or equivalents. Reagents include any compound that facilitates the analysis of multicellular structures such as organoids. Exemplary reagents include, among other things, labels, solidifying agents, and clarifying agents. Labels may include dyes (e.g., visible stains and / or photoluminescent dyes). Photoluminescent dyes are any substance that emits light in response to irradiation accompanied by electromagnetic radiation, such as excitation light.

[0088] Each reservoir 105 may have an open top to facilitate the introduction and removal of fluid 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 cultivation in the incubator. The lid may have a flange that overlaps the upper region of each reservoir perpendicularly and optionally configured to restrict the lateral movement of the lid when covering the reservoir without creating a tight fit. In some embodiments, the lid may be a cap that forms a liquid-tight seal over the top of one or more reservoirs 105.

[0089] The container 102 may contain at least one module 109 in one or more of the container's compartments. Figure 1A shows a module 109 located within the culture chamber 103, with possible alternative locations for the same module indicated by the illusion lines. More specifically, each module 109 may be located, among other things, within the culture chamber 103, the reservoir 105, or the slot 107. Thus, each module 109 may be located within or adjacent to the culture chamber 103. Each module 109 may be contained within the culture vessel 102, or may exist in a set of functionally different modules 109 located outside the culture vessel 102 and configured to be selectively placed inside the culture vessel 102 by the user. Each module 109 may independently be an active electrical / magnetic module (e.g., an electrode module, an actuator module, a sensor module, a pump module, and / or a lighting (light-emitting) module) or a passive module (e.g., a transparent interface module, a scaffolding module, or a dummy module), as further described below.

[0090] The culture system 100 may also include an incubator 110 for containing the container array 101 (see Figure 1). The incubator may be temperature-controlled to a suitable culture temperature for the multicellular structure 104, such as at least 25°C, 30°C, or 35°C (see also Figure 1A). The humidity and / or atmosphere inside the incubator 110 may also be controlled to promote the growth and development of the multicellular structure 104.

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

[0092] The control circuit 116 of the culture system 100 powers and / or operates any suitable device of the culture system. For example, the control circuit 116 may control the fluid transport system 111, the detection system 112, and / or the conveyor 113, respectively, via wired or wireless communication, which may be unidirectional or bidirectional. Each module 109 held by the container array 101 may also be wirelessly controlled by the control circuit 116, as indicated by the dashed arrows in 117. This wireless control is advantageous because it simplifies the structure of the container array 101, allows the container array to be functionally customized as needed by introducing appropriate modules, and eliminates the need for wires or electrical conductors that extend into the culture vessels and provide pathways for microbial contamination.

[0093] Figure 2 shows an exemplary container array 201 relating to the culture system 100 of Figure 1. The container array comprises a plurality of container strips 218 (synonymously referred to as container assemblies), each containing a row of culture vessels 202 that are connected to one another, for example, via a common (shared) housing. A container strip may have any preferred number of culture vessels 202, such as at least two, three, four, or more. Each container strip 218 is received and held by a frame 219, optionally forming a row of container strips 218. Each container strip 218 may be installed as a unit in individual receiving sites of the frame 219, for example, during manufacturing or by the user. In the embodiment depicted, the frame 219 has a row of eight receiving sites for receiving a corresponding number of container strips 218, but in other embodiments, the frame may be configured to receive at least two, three, or more container strips 218 in a corresponding number of receiving sites. Each container strip 218 may be individually removable from the frame 219 to allow for the manipulation, processing, and / or analysis of the container strip (and / or its contents) independently of the other container strips 218 in the container array 201.

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

[0095] Figure 4 shows the culture vessel 102 of Figure 1A and a set of module 420 of functionally distinct modules 409a-409c that may be contained within the culture vessel 102. The set of module 420 may consist of any preferred number of functionally distinct modules, such as at least two, three, four, or more. Each module in the set of module 420 may have any preferred combination of properties, as described above with respect to module 109 or elsewhere in this specification (e.g., in Sections I, III, IV, and VI). Two or more modules of module set 420 may be interchangeably positioned within the culture vessel 102, for example, in the same compartment, as illustrated by illusory arrows relating to the placement of modules 409b and 409c in slot 107, and / or at least one module of module set 420 may be interchangeably positioned within each of two or more compartments of the culture vessel 102, as illustrated by a pair of illusory arrows relating to module 409a in culture chamber 103 or reservoir 105.

[0096] Figure 5 shows an exemplary culture vessel 502 and exemplary control circuit 516 relating to the culture system 100 of Figure 1. The culture vessel 502 is shown in a schematic side view and includes a culture chamber 503 containing a multicellular structure 504, i.e., organoids 521 and associated scaffolds 522 for assisting the formation and / or growth of the organoids. The scaffolds 522 are attached to the bottom wall of the culture chamber 503 in Figure 5, but in other embodiments they may be attached to any preferred side wall or top wall of the culture chamber. At least a pair of reservoirs 505a, 505b are located across the culture chamber 503 and communicate with the culture chamber via separate channels 506a, 506b. Each reservoir is located "vertically above" the culture chamber, meaning that a vertical line extends through the reservoir and the culture chamber. Each reservoir 505a, 505b holds a separate culture medium 523a, 523b, which may have the same or different compositions. The culture chamber 503 also holds a culture medium, which may be supplied at least partially 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, as the slot can 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 at least 50% or 100% greater. 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 the user. In some cases, the user may select a slot module 509a for installation in the slot 507 from a set of functionally different slot modules (see, for example, Figure 4). The ability to swap slot modules of different functions allows the culture vessel 502 to be adapted to different culture / test configurations for various types of organoids or other multicellular structures. In some cases, a dummy module may be placed in slot 507 to cover the opening 508 if the slot module is not being used in the slot.

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

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

[0100] The culture chamber 503 may contain at least one chamber module 509b. The chamber module may be installed in the receptacle 524 via its top, bottom, or side, and the receptacle 524 may be converted into the culture chamber 503 by closing off at least partially the top, bottom, or side of the receptacle. In some cases, if the chamber module 509b is sufficiently large, the chamber module may remain confined within the culture chamber 503 until the culture chamber is opened and / or dismantled by removing its wall portion, and until such dismantling occurs.

[0101] The control circuit 516 is configured to wirelessly control the slot module 509a and / or chamber module 509b, as shown in 517, if one or both are electrical / magnetic modules (see Section III). The control circuit may include one or more antennas 525 to transmit / receive power and / or data from and to each module 509a, 509b, which are electrical modules, if present, using near-field radiation. One or more coils 526 of the control circuit may be used to generate a magnetic field to drive the movement of at least the magnetic portion of each module 509a, 509b, if present. The control circuit 516 may also include 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 equivalent).

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

[0103] Figure 6 is a flowchart 630 of exemplary steps 631a–631j, which can be carried out in any preferred order and combination to provide a method for culturing multicellular structures. The method may be carried out using any preferred system, device, cell, and scaffold of this disclosure.

[0104] The scaffold may be placed inside the receptacle or culture chamber of the culture vessel in step 631a. The scaffold may be placed by forming it inside the receptacle or culture chamber, for example by 3D printing, or by installing a pre-formed scaffold inside the receptacle or culture chamber. In some embodiments, the scaffold may be attached to a scaffold module that can be installed inside the slot of 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 intended to generate organized multicellular structures through differentiation, division, migration, etc. Otherwise, cells may be introduced into the receptacle or culture chamber as a pre-formed multicellular structure (e.g., organism, tissue explant, tumor, organoid, or equivalent). Cells may be introduced during step 631a, or 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 preferred number of one or more modules may be installed during the 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 that may have an open side, and at least partially closing the open side. For example, the open side may be covered by joining a sealing member to the receptacle on the open side (e.g., on the open bottom side of the receptacle), or by placing an inserter inside the shell containing the receptacle. In either case, the chamber may be formed cooperatively using the receptacle and the sealing member or inserter.

[0108] The module may be placed in the container slot 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 that includes a pre-formed scaffold. In some cases, steps 631b and 631e may be performed together using a module that includes cells.

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

[0110] The container and its contents may be cultured in step 631 g. The culture may be carried out at a suitable temperature in a suitable gaseous atmosphere, in particular for any suitable duration such as at least 1, 2, 3, 4, or 5 days, or at least 1, 2, or 3 weeks.

[0111] If present, each electrical / magnetic module in the culture vessel may be wirelessly controlled by a control circuit in step 631h. This control may include transmitting power, force, and / or data to the module in order to drive the movement of the module or at least a portion of it, and / or to control the 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 during step 631i. Step 631i may be performed in response to steps 631g and / or 631h.

[0113] In step 631j, the first module contained in the container may be removed and replaced with a second module. The first module may be removed from the slot of the container and replaced with a second module in the same slot. The first and second modules may be functionally different from each other. After the replacement of the first module with the second module, steps 631h and 631i may be repeated.

[0114] Figure 7 shows flowchart 731ab, 731d, 731f, 731g, and 731i of exemplary steps 731ab, 731d, 731f, 731g, and 731i of flowchart 630 in Figure 6, which can be optionally carried out in any preferred order and combination, by adding one or more steps from flowchart 630 in Figure 6. The method may be carried out using any preferred system, device, cell, and scaffold 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 placed inside the shell of the container in step 731ab. More specifically, the scaffold and / or cells may be placed inside a receptacle formed by a portion of the shell.

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

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

[0118] The culture vessel and its contents may be cultured in step 731g. The culture may be carried out at a suitable temperature, in a suitable gaseous atmosphere, and for any suitable duration, as described above with respect to step 631g (see Figure 6).

[0119] The methods shown in Figures 6 and 7 may be carried out using an array of culture vessels. Therefore, each step of this method may be performed on the array of culture vessels as a group, or on individual culture vessels, as appropriate. III. Electrical / Magnetic Modules

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

[0121] Figure 8 shows an exemplary electrode module 809 (i.e., an electrical / magnetic embodiment of module 109), and also shows the power antenna 825a and communication antenna 825b of the control circuit 816. Antennas 825a and 825b are configured to transmit power to the electrode module 809 and operate it, respectively.

[0122] The electrode module 809 comprises a housing 832 supporting an electrode interface 833. The electrode interface may be located on any preferred side of the housing 832, such as the bottom side as shown, or its lateral side (for example, if the electrode module 809 is contained within a culture chamber). The electrode interface 833 may include any preferred number of electrodes of any preferred shape. For example, the electrode interface may have a pair of plate electrodes 834a configured to touch the surface of a multicellular structure within a culture chamber and / or a pair of pin electrodes 834b configured to penetrate and extend into the multicellular structure. The electrode interface 833 may be used to electrically stimulate the multicellular structure and / or to sense the electrical properties of the multicellular structure from the outside or inside. Exemplary uses of electrode module 809 include, among other things, 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 influence network formation. Other exemplary uses of electrode module 809 include sensing the electrical activity of any suitable cell / tissue such as neurons, smooth muscle, cardiomyocytes, or skeletal muscle.

[0123] The housing 832 may include any suitable electronic circuitry to enable wireless power supply to, communication with, and / or control of the electrode module 809. This circuitry may include a power receiver 835 having an antenna for receiving power wirelessly transmitted from the power antenna 825a of the control circuit 816. The circuitry may also include an internal communication antenna 836 to receive signals from and / or transmit signals to the external communication antenna 825b of the control circuit 816 in order 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 used. 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] Figure 9 shows an exemplary sensor module 909 (i.e., an electrical / magnetic embodiment of module 109) and also shows the communication antenna 925 of the control circuit 916. The antenna 925 is configured to transmit power to the sensor module 909 to operate it. Therefore, a separate power antenna and power receiver may not be necessary (compare with Figure 8).

[0125] The sensor module 909 comprises a housing 932 supporting a sensor interface 940. The sensor interface may be located on any preferred side of the housing 932, such as the bottom side as shown, or its lateral side (e.g., when the sensor module 909 is contained within a culture chamber). The sensor interface 940 may be configured to sense and measure any preferred physical or chemical parameter, such as temperature, motion, electrical parameters (e.g., potential, current, impedance, etc.), electric / magnetic fields (e.g., using a Hall effect sensor), pH, chemical potential, oxygen or carbon dioxide concentration, chemical substances (e.g., using an electrochemical sensor). The sensor module 909 may be preferred when remote sensing is difficult or impossible, or when it is advantageous to establish direct contact between the sensor interface 940 and the multicellular structure to be analyzed.

[0126] The housing 932 may include any suitable electronic circuitry to enable wireless power supply to, communication with, and / or control of the sensor module 909. This circuitry may include an internal communication antenna 936 to receive signals from and / or transmit signals to the external communication antenna 925 of the control circuit 916 in order to power and operate the sensor module 909. Any suitable communication protocol, such as at least one near-field communication (NFC) protocol, may be used. 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] Figure 10 shows an exemplary pump module 1009 (i.e., an electrical / magnetic embodiment of module 109) and also shows the power antenna 1025 of the control circuit 1016. The power antenna 1025 is configured to transmit power to the pump module 1009. A separate communication antenna may or may not be present in the control circuit 1016 (compare with Figure 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 around the pump module 1009. The operation of the pump 1044 is to draw fluid into nozzle 1045a (acting as an inlet) and push fluid out of nozzle 1045b, or vice versa if the pump is driven in the reverse direction. This fluid movement can generate turbulence, among other things, to enhance the flow of the culture medium in the culture vessel and / or to stimulate organoid development. The use of the pump module 1009 may be preferred when gravity-driven flow is not applicable or is inefficient (for example, due to the properties of the culture medium).

[0129] The enclosure 1032 may include any suitable electronic circuitry to enable wireless power reception from the control circuit 1016 and operation of the pump 1044. This circuitry may include a power receiver 1035 to receive power wirelessly transmitted 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 drive unit 1046.

[0130] Figure 11 shows an exemplary culture system 1100, including a pair of pump modules 1009a, 1009b contained within a culture vessel 1102 (see also Figure 10). Vessel 1102 includes a culture chamber 1103 containing an organoid 1121. A slot 1107 is located vertically above the culture chamber 1103, housing three compartments: a pair of reservoirs 1105a, 1105b holding individual culture media 1123a, 1123b, and a dummy module 1109 (an embodiment of module 109 in Figure 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 of the control circuit 1016, respectively. Pump 1009a drives culture medium 1123a from reservoir 1105a through channel 1106c into culture chamber 1103. Pump 1009b drives culture medium from culture chamber 1103 through channel 1106d 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 the medium from reservoir 1105b back to reservoir 1105a, or this may be done via gravity-driven flow through channels 1106a and 1106b.

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

[0133] The electrode module 809 may be located within the slot 1107, with both pin electrodes 834b extending into the organoid 1221 (see also Figure 8). However, as discussed below, the mechanical stimulation of the organoid 1221 provided by the 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 to facilitate adhesion to the organoid 1221. The magnets 1247 of magnetic modules 1209a, 1209b each have a North Pole (N) and a South Pole (S), and their magnetic axes extend through both poles. The magnetic modules 1209a, 1209b may be arranged within the cell chamber 1103 such that the magnetic axes of the magnetic modules are coaxial with each other and antiparallel as shown, creating magnetic repulsion, or parallel, creating magnetic attraction.

[0135] The control circuit 1216 includes at least one coil (equivalent to a pair of fixed coils 1226a, 1226b) that can be electrically energized to create one or more additional magnetic fields. The additional magnetic fields effectively enhance or reduce the attractive or repulsive forces between 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 which can be oriented parallel to the magnetic axes of magnetic modules 1209a, 1209. Figure 13 illustrates the energization of both coils 1226a, 1226b, indicated by current arrows 1348, and the generation of indicated magnetic field polarities in the coils. The magnetic repulsion between coil 1226a and magnetic module 1209a, and between coil 1226b and magnetic module 1209b, drives the mutually directed movement of the magnetic modules, indicated by the motion arrows in 1349, which applies compression to organoid 1221. In other cases, energizing coils 1226a and 1226b may drive magnetic modules 1209a and 1209b further, which may apply tension to the organoid if they are attached to both modules. In 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 with respect to certain types of organoids, such as bone organoids and muscle organoids, which may require this stress for proper development.

[0136] Figures 14 and 15 schematically illustrate how the magnetic drive mechanism of Figures 12 and 13 may be implemented in a culture system 1400 including a row of culture vessels 1402a-1402c. (The positions of the culture vessels are roughly indicated by dashed arrows, but the vessels themselves are omitted for the sake of simplicity in the illustration.) Each culture vessel 1402a-1402c contains individual organoids 1421a-1421c sandwiched between pairs of magnetic modules 1209a, 1209b, as described above with respect to a single culture vessel 1102. The control circuit 1416 provides a series of electrically energized coils 1426a-1426d arranged along the same lines as each pair of magnetic modules 1209a, 1209b, including the vessels 1402a-1402c, the organoids 1421a-1421c, and the magnetic modules 1209a, 1209b. The coil axes defined by coils 1426a-1426d each extend along the rows of containers 1402a-1402c.

[0137] The positions of magnetic modules 1209a and 1209b are shown in Figure 14 without electrical energization of coils 1426a-1426d, and in Figure 15 with electrical energization. Electrical energization is indicated by the arrow at 1448 in Figure 15. As described above with respect to the single culture vessel in Figures 12 and 13, proper electrical energization of coils 1426a-1426d drives each pair of magnetic modules 1209a and 1209b linearly toward each other along the line of vessel 1402a-1402c, which allows organoids 1421a-1421c to be mechanically stimulated in a synchronous manner.

[0138] Figure 16 is a schematic top view of culture system 1600, having different configurations of electrically energized coils 1626 for driving the movement of magnetic modules 1209a and 1209b as shown in Figures 14 and 15. (Only a subset of coils 1626 are identified using numerical identifiers.) Culture system 1600 includes a row of containers 1602a-1602c. (The positions of the containers are roughly indicated using dashed arrows, but the containers themselves are omitted for the sake of simplicity in the illustration.) Each container 1602a-1602c contains individual organoids 1602a-1602c sandwiched between pairs of magnetic modules 1209a and 1209b, as described above with respect to culture system 1400. However, the magnetic axes of each pair of magnetic modules 1209a and 1209b are oriented orthogonal to the row of containers 1602a-1602c, such as horizontally, as shown. The control circuit 1616 provides at least one row or a pair of rows of coils 1626. Each coil 1626 may define a coil axis 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 containers 1602a-1602c, and mechanically stimulates the organoids 1621a-1621c.

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

[0140] Exemplary light sources include light-emitting diodes, lasers, or equivalents. The light source may also include any suitable optical system for directing or focusing the light. For example, the light source may have waveguides, such as optical fibers, to direct light from the optical module 1709 onto the surface of the multicellular structure and / or to extend into the multicellular structure and illuminate it from the inside.

[0141] The optical module 1709 may be used to facilitate imaging of multicellular structures contained within the culture chamber of a culture vessel. For example, when the optical module is appropriately positioned within the culture vessel, such as in its reservoir, slot, or culture chamber, it may provide bright-field or dark-field illumination of the multicellular structure. 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 power source and without interaction with control circuits. Each passive module may be configured to be installed in a specific compartment of a vessel, or alternatively, in each of two or more compartments of the vessel.

[0143] Figure 18 is a somewhat schematic diagram of an exemplary scaffold module 1809, which is an embodiment of module 109 of Figure 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, for example by 3D printing, or it may be formed first and then attached to the body 1851. In either case, the scaffold 1822 may be mounted on any preferred side of the body 1851, such as the bottom side, top side, or lateral side. The scaffold module 1809 may be located in a slot of a culture vessel, for example, during the manufacture of the culture vessel or installed in a slot by the user. In some cases, the scaffold module may be an inserter that forms a reservoir of one or more culture vessels, as further described below in Section V. Scaffolding modules may be used to mount and support scaffolding, as well as to keep the scaffolding in place, and to enable improved handling and scaffolding addition and removal.

[0144] Figure 19 shows an exemplary permeable interface module 1909, which is an embodiment of module 109 of Figure 1A. The permeable interface module is cross-sectioned in Figure 19 to expose its internal structure. The permeable interface module 1909 has a hollow body 1951 that defines a cavity 1952 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, creating a permeable interface 1957 (e.g., a permeable wall) on any preferred side of the body 1951, such as the bottom, top, or side. The permeable interface 1957 optionally and selectively allows fluid and / or small molecules to pass in and out of the cavity 1952 through 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 equivalent. The culture medium may have a different phase, composition, and / or chemical potential from the culture medium present in the culture chamber of the container. The permeable interface 1957 may be positioned in physical contact with the multicellular structure and / or culture medium in the container. In one example, the permeable interface module 1909 may contain air (or other gas) within the cavity 1952, creating a gas / liquid interface in the permeable member 1956, enabling the culture of lung organoids in the culture chamber of the culture vessel. In another example, the permeable interface module 1909 may contain any suitable chemical substance, which can be discharged into the culture chamber of the culture vessel through the permeable member 1956.

[0146] Figure 20 shows a permeable interface module 1909 located within a slot 1107 of the container 1102 (see also Figure 11), adjacent to the opening 1108 and operationally positioned across the lung organoid 2021. The cavity 1952 is filled with gas such that a 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. The dummy module may be a substitute. Like any of the modules of this disclosure, the dummy module may function to reduce the fluid holding capacity of the vessel and / or to block or seal a slot across the culture chamber. However, the dummy module may also provide a surface shape (concave or convex), surface chemistry / texture (e.g., hydrophilicity, microstructure, etc.), and / or a functional surface that is advantageous with respect to the culture protocol. V. Container Assembly

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

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

[0150] The container strip 2118 forms rows of at least two, three, or more culture vessels, such as four culture vessels 2102a-2102d in the embodiment described. Each culture vessel 2102a-2102d includes a separate culture chamber 2103, which is formed by the lower region of the culture vessel.

[0151] Figure 22 shows the container strip 2118 being removed from frame 2119 to enable imaging of the multicellular structure contained within the culture chamber 2103. Removal of the container strip 2118 from frame 2119 allows the container strip to be properly positioned relative to the imaging system 2112 (or other detection system) without interference from frame 2119 or other container 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 a smooth inner and outer surface that is optionally planar to minimize scattering of optical radiation. In the embodiments described, each culture chamber 2013 has a bottom optical window 2159a and a pair of lateral optical windows 2159b, 2159c arranged opposite each other across the culture chamber 2103 (see also Figures 24, 26, and 30).

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

[0154] The container strip 2118 provides separate pairs of reservoirs 2105a, 2105b and separate slots 2107, located across the culture chambers 2013 of each container 2102a-2102d (see Figures 21 and 22). Slot modules 2109 may be located within the slots 2107 and may be any of the electrical / magnetic or passive modules disclosed herein (see, for example, Sections I, III, and IV).

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

[0156] The shell 2162 may be internally divided into multiple sections, such as sections 2165a-2165d. Sections 2165a-2165d may be formed integrally with each other. Sections 2165a-2165d may be directly attached to each other, or they may be separated from each other using separate spacer regions 2166 located between each adjacent pair of sections.

[0157] Each section 2165a–2165d of the shell 2162 includes a lower region that forms a receptacle 2167 and an upper region that forms a receptive space 2168 for one of the inserters 2163 (see Figures 23, 26, and 28–30). Each culture chamber 2103 is cooperatively formed using the shell 2162 and one of the inserters 2163, which may capture a module (in the embodiment depicted, a pair of magnetic modules 1209a, 1209b, etc. (see Figure 26)) within the culture chamber. The receptacle 2167 provides the lateral wall of the culture chamber 2103, and one of the inserters 2163 provides the upper wall portion of the culture chamber. A scaffold for the organoid may be located within the receptacle 2167 of each inserter 2163, for example, by being formed or installed within the inserter 2163 before it is assembled with the shell 2162 (e.g., by being attached to the bottom or side walls of the receptacle). In other cases, the scaffold may be located on the bottom side of the inserter 2163, for example, by being 3D printed on it before the inserter is assembled with the shell 2162, or by being attached after the scaffold is formed. In yet 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. Therefore, the external 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, forming a shoulder 2169 at the bottom of the receiving space 2168 to support the inserter 2163 and prevent further downward advance into the section (see Figures 26, 29, and 30).

[0159] Each inserter 2163 may be secured within the shell 2162 by a snap-fitting mechanism that engages when the inserter is seated within the shell (see Figures 27 and 31-33). The inserters may define projections 2170 that are received within individual openings 2171 defined by the lateral walls of the shell 2162, or the inserters may define openings and the shell may define projections.

[0160] Each inserter 2163 defines various openings at its bottom. The openings at the bottom of each reservoir 2105a, 2105b include at least one equal channel, a pair of channels 2106a, 2106b (see Figure 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 Figure 26). The inserter also defines an opening 2108 at the bottom end of slot 2107, providing fluid communication between slot 2107 and the culture chamber 2103 (see Figures 26, 33, 34).

[0161] Figure 35 shows a lid assembly 2172 for a container array 2101 (see also Figure 21). The lid assembly 2172 comprises a series of lids 2164 for covering a corresponding number of container strips 2118 held by a frame 2119 (see also Figure 26). Each lid 2164 may be fitted onto the top of a container strip (e.g., the top of a shell 2162) and may have a peripheral flange configured to protrude downward, overlap the container strip perpendicularly at its upper edge, and horizontally surround it. Maintaining sterility can be a major issue throughout the entire culture and testing process, which can take up to several months. Therefore, a lid assembly 2172 would be advantageous for blocking the entry of contaminating microorganisms through the open tops of the reservoirs 2105a, 2015b and slots 2107 of each container 2102a-2102d of each container strip 2118. The lid assembly 2172 allows all 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 removable (e.g., via a weak adhesive, interlocking, or snap-fitting mechanism). This configuration allows for the easy removal of any single container strip 2118, including its lid 2164, from other container strips 2118 and their lids 2164 of the container array 2101, for imaging or other processing, etc. In other embodiments, the carrier 2173 may be omitted, and the lids 2164 may be formed integrally with each other and configured to be removable by breaking a brittle connection that joins adjacent pairs of lids 2164 together.

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

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

[0164] Figure 39 shows yet another exemplary inserter 3963 for placement within the shell 2162 (see also Figure 23). The inserter 3963 forms four reservoirs 3905a-3905d and a central slot 3907 located between pairs of reservoirs (compare with Figure 33). The inserter 3963 may be advantageous when multicellular structures are being fed different culture media from both the inside and outside.

[0165] Figure 40 shows yet another exemplary inserter 4063 for placement within shell 2162 (see also Figure 23). Inserter 4063, like inserter 3963, forms four reservoirs 4005a–4005d, but lacks a central slot located midway between pairs of reservoirs (compare with Figure 39). Inserter 4063 has more than two reservoirs and combines the potential advantage of centrally locating the channel across multicellular structures. VI. Selected Aspects

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

[0167] Paragraph A1. A system for culturing multicellular structures such as organoids, comprising: (a) a container including a culture chamber for containing a multicellular structure (such as an organoid); (b) an electrical / magnetic module optionally configured to be coupled to and / or located within the container in or adjacent to the culture chamber; and (c) a control circuit configured to wirelessly power and / or operate the electrical / magnetic module, wherein the electrical / magnetic module optionally detachably coupled to and / or located within the container, and optionally the electrical / magnetic module can be received in each of two or more compartments of the container, the two or more compartments optionally selected from a culture chamber, one or more reservoirs, and / or slots.

[0168] Paragraph A2. The control circuit is configured to wirelessly power and / or operate an electrical / magnetic module using near-field radiation, as described in paragraph A1.

[0169] Paragraph A3. The system described in Paragraph A2, wherein the control circuit is configured to wirelessly transmit power to an electrical / magnetic module via inductive or capacitive coupling.

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

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

[0172] Paragraph A6. The system according to Paragraph A5, wherein the electrical / magnetic module is a first module, and the magnets are a first magnet, further comprising a second module containing a second magnet, the first and second modules being contained in or configured to be contained in a culture chamber at the same time, and the control circuit is configured to drive the movement of the first and second magnets toward and away from each other within the culture chamber, at will.

[0173] Paragraph A7. An electrical / magnetic module is a system described in any of paragraphs A1-A6, including a sensor.

[0174] Paragraph A8. An electrical / magnetic module is a system described in Paragraph A7, which includes a chemical sensor, an electrical sensor, an optical sensor, and / or a temperature sensor.

[0175] Paragraph A9. An electrical / magnetic module is a system described in any of paragraphs A1-A8, including electrodes.

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

[0177] Paragraph A11. An electrical / magnetic module is a system described in any of paragraphs A1-A10, including a light source.

[0178] Paragraph A12. The system described in 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 described in any of paragraphs A1-A12, including a pump, which is configured to drive fluid flow in and / or out of the culture chamber.

[0180] Paragraph A14. The electrical / magnetic module is located in or configured to be located in 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, as described in any of paragraphs A1-A13.

[0181] Paragraph A15. The system according to Paragraph A14, wherein the vessel comprises two or more reservoirs that are in fluid communication with the culture chamber, optionally, the slots are located vertically above the culture chamber, optionally between at least one pair of the two or more reservoirs, optionally, each of the two or more reservoirs is 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, 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. The system described in paragraph A14 or A15, wherein the electrical / magnetic modules are contained within a set of two or more modules, configured to perform different functions from each other and to be coupled to and / or located within a container, and are optionally interchangeably positioned within the same compartment of a container such as the same reservoir, culture chamber, and / or slot.

[0183] Paragraph A17. Optionally, each container is positioned across a culture chamber (optionally, vertically above the culture chamber) and optionally includes at least one or more reservoirs sharing a wall with the culture chamber, optionally the two or more reservoirs being formed integrally with each other and / or by identical inserters, optionally an electrical / magnetic module being positioned vertically between the upper side of the two or more reservoirs and the bottom of the culture chamber, and / or the system further optionally includes 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 according to any of paragraphs A1-A17, further comprising a container assembly that is interconnected and forms a row of containers, each container in the row of containers comprising an individual culture chamber for containing a multicellular structure (such as an organoid), optionally the containers in the row of containers being substantially identical to each other, and optionally an electrical / magnetic module being movable between the containers in the row of containers.

[0185] Paragraph A19. The container assembly is the system described in Paragraph A18, having a length corresponding to the length or width of the standard microplate occupancy area.

[0186] Paragraph A20. The system according to paragraph A18 or A19, further comprising a container array, which includes a container assembly, further comprising a container array, wherein each of the two or three rows of containers comprises a separate culture chamber for containing a multicellular structure.

[0187] Paragraph A21. A system according to 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 comprising a plurality of corresponding shells, where the plurality of container assemblies are substantially identical to each other.

[0188] Paragraph A22. The container is a system according to any of paragraphs A1-A21, including an optical window formed by the wall of the culture chamber.

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

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

[0191] Paragraph B1. A method for culturing a multicellular structure such as an organoid, comprising: (a) a step of containing the multicellular structure in a culture chamber of a container, wherein an electrical / magnetic module is optionally removablely coupled to the container and / or located within the culture chamber or adjacent thereto; and (b) a step of wirelessly powering / operating the electrical / magnetic module using a control circuit, wherein the electrical / magnetic module is optionally removablely coupled to the container and / or located within it, and optionally the electrical / magnetic module is located at two or more points in the container. The container can be accommodated in each of the compartments above it, and optionally, two or more compartments are selected from a culture chamber, one or more reservoirs, and / or slots, and optionally, the multicellular structure is an organoid with a diameter of at least 0.2, 0.5, 1, or 2 mm, and is formed / grown within the culture chamber without connecting the container to an external source of liquid (e.g., culture medium) (e.g., via tubes and / or tubing), and / or without electrically connecting the container to wires or other electrical conductors, method.

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

[0193] Paragraph B3. The method according to paragraph B1 or B2, wherein the power supply / operation step is carried out at least in part via inductive or capacitive coupling of the control circuit and the electrical / magnetic module to each other.

[0194] Paragraph B4. The method according to any of paragraphs B1-B3, wherein the step of supplying power / operating includes the step of communicating wirelessly with the electrical / magnetic module using near-field radiation.

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

[0196] Paragraph B6. The method according to any of paragraphs B1-B5, wherein the electrical / magnetic module includes a magnet, and the steps of powering / operating it include driving the movement of the magnet within the culture chamber using a magnetic field created by a control circuit.

[0197] Paragraph B7. The method according to paragraph B6, wherein each culture chamber contains a pair of electrical / magnetic modules, each containing a magnet, and the step of powering / operating the electrical / magnetic modules includes the step of driving them toward and away from each other within the culture chamber, as desired, toward and away from each other.

[0198] Paragraph B8. The method according to any of paragraphs B1-B7, wherein the power supply / operation step includes using sensors of an electrical / magnetic module to sense the properties of a multicellular structure (e.g., located within a culture chamber) and / or the culture medium in contact with the multicellular structure.

[0199] Paragraph B9. The power supply / operation step is the method according to any of paragraphs B1-B8, wherein the step of electrically stimulating a multicellular structure is included.

[0200] Paragraph B10. The method according to any of paragraphs B1-B9, wherein the power supply / operation step includes the step of driving fluid flow in and / or out of the culture chamber.

[0201] Paragraph B11. The electrical / magnetic module is located within the culture chamber, as described in any of paragraphs B1-B10.

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

[0203] Paragraph B13. The method described in Paragraph B12, wherein the electrical / magnetic module is located within a slot defined by the container.

[0204] Paragraph B14. The method according to any of paragraphs B1-B13, further comprising the step of collecting data related to the multicellular structure while the multicellular structure remains in the culture chamber.

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

[0206] Paragraph B16. The step of collecting data is carried out using the module's sensors, as described in paragraph B14 or B15.

[0207] Paragraph B17. The method according to any of paragraphs B1-B16, further comprising the step of placing an electrical / magnetic module within a compartment of a container.

[0208] Paragraph B18. The method according to paragraph B17, wherein the placement step includes the step of capturing the electrical / magnetic module in the culture chamber.

[0209] Paragraph B19. The method according to paragraph B17, wherein the placement step is inside the container, but optionally includes the step of placing the electrical / magnetic module outside the culture chamber, in the reservoir or slot of the container.

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

[0211] Paragraph B21. The method according to paragraph B20, further comprising the step of selecting two or more functionally different modules from a set, and the placement step comprising, for each selected module, the step of combining the selected modules into a container and / or placing the selected modules within the container.

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

[0213] Paragraph B23. The method described is the method described in any of paragraphs B1-B22, performed using the system described in any of paragraphs A1-A24.

[0214] Paragraph C1. A device for culturing multicellular structures such as organoids, comprising (a) a shell having an open top at the option of any choice, and (b) an inserter comprising at least one reservoir or two or more reservoirs, wherein the inserter is configured to be optionally received into the shell via the open top so that the shell and the inserter cooperate to form a culture chamber for a multicellular structure, the culture chamber being located below the two or more reservoirs (optionally, vertically downward) and optionally in fluid communication with each of the at least one reservoir or two or more reservoirs via separate channels defined by the inserter.

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

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

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

[0218] Paragraph C5. The scaffolding is the device described in Paragraph C4, which is attached to the wall of the shell.

[0219] Paragraph C6. The device according to paragraph C4, further comprising a module which is part of the container and provides a scaffold, the module being configured to be coupled (optionally, detachably) to and / or located within a compartment of the container, such as a slot defined by the inserter.

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

[0221] Paragraph C8. The device according to any of paragraphs C1-C7, wherein the shell comprises a division having an upper region defining a receptive space and a lower region forming a receptacle, the inserter is configured to be received into the receptive space, and the receptacle is configured to cooperatively form a culture chamber together with the inserter.

[0222] Paragraph C9. The inserter is configured to form a liquid-tight seal with multiple side walls and / or bottom walls of the upper region of the section, as described in paragraph C8.

[0223] Paragraph C10. The device described in paragraph C9, comprising a body and a gasket attached to the body, wherein the gasket is configured to engage with each of the side walls and / or bottom walls of a plurality of side walls to form a liquid-tight seal.

[0224] Paragraph C11. The gasket is molded onto the body of the device as described in Paragraph C10.

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

[0226] Paragraph C13. The device according to any of paragraphs C1-C12, comprising a plurality of inserters, the shell forming a row of compartments, each configured to receive an individual inserter of the plurality of inserters, and each compartment and individual inserter cooperatively forming a culture chamber for a multicellular structure.

[0227] Paragraph C14. The divisions in the column of divisions are formed integrally with each other as a single part, as described in Paragraph C13.

[0228] Paragraph C15. The device described in paragraph C13 or C14, further comprising a lid configured to be mounted on the shell in order to cover the columns of divisions.

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

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

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

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

[0233] Paragraph C20. The device according to any of paragraphs C1-C19, wherein the shell and inserter each comprise one or more modules, each configured to form a portion of the same vessel and each to be coupled to and / or located within the vessel, and optionally further comprising two or more sets of functionally different modules, such as a set comprising at least one or at least two electrical / magnetic modules and / or at least one or at least two passive modules, and optionally at least two of the functionally different modules to be interchangeably installed within the same compartment of the vessel (e.g., the same reservoir, culture chamber, or slot).

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

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

[0236] Paragraph D3. The method according to paragraph D1 or D2, wherein the culture chamber is in fluid communication with each of the two or more reservoirs via a channel defined by an inserter.

[0237] Paragraph D4. The method according to any of paragraphs D1-D3, further comprising the step of placing a scaffold within a shell, wherein the scaffold is configured to facilitate organoid formation within a culture chamber, and optionally, cells of a multicellular structure are embedded within the scaffold.

[0238] Paragraph D5. The method of Paragraph D4, wherein the disposing step optionally comprises forming a scaffold within the shell prior to installing an inserter.

[0239] Paragraph D6. The method of Paragraph D4, wherein the disposing step comprises placing a pre-formed scaffold into the shell.

[0240] Paragraph D7. The method of Paragraph D6, wherein the step of placing the pre-formed scaffold into the shell is performed before installing the inserter.

[0241] Paragraph D8. The method of Paragraph D6, wherein the step of placing the pre-formed scaffold into the shell is performed by or after installing the inserter, and optionally, the pre-formed scaffold is already attached to a bottom side of the inserter before installing the inserter.

[0242] Paragraph D9. The method of any one of Paragraphs D1-D8, wherein the shell comprises a row of compartments, the installing step comprises installing at least two inserters into the row of compartments to form two or more separate culture chambers, and the step of culturing the multicellular structure comprises culturing a separate organoid in each of the two or more separate culture chambers.

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

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

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

[0246] Paragraph E1. A system for culturing multicellular structures such as organoids, comprising: (a) a container comprising a culture chamber for containing the multicellular structure, at least one reservoir or at least two or more reservoirs and an optional slot, each being in fluid communication with the culture chamber; and (b) two or more modules having different functions from each other, configured to be exchangeably 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 one of Paragraphs E1 to E3, wherein the two or more modules comprise 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 liquid in the culture chamber and liquid or gas in the first module.

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

[0252] Paragraph E7. The system of Paragraph E6, wherein the at least one module comprises 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 comprise an electronic device.

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

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

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

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

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

[0259] Paragraph F5. The method according to any of paragraphs F1-F4, wherein the modules of 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 according to any of paragraphs F1-F5, wherein at least one of the first and second modules includes an electrode that is in contact with a multicellular structure and / or liquid in a culture chamber.

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

[0262] Paragraph F8. The container is provided by the device described in any of paragraphs C1-C20, according to the method described in any of paragraphs F1-F7.

[0263] Paragraph G1. Organoid culture vessels that do not require tubes and wires to be connected to the vessel itself.

[0264] Paragraph H1. A method for culturing organoids, comprising the step of forming / growing an organoid (e.g., a large organoid) in a container without connecting the container to an external source of liquid (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 / magnetic device and / or an electrical conductor electrically connected to a control circuit).

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

Claims

1. A device for culturing a multicellular structure, wherein the device is A shell with an open top, An inserter comprising two or more reservoirs, wherein the inserter is configured to be received into the shell via an open top so that the shell and the inserter cooperate to form a culture chamber for the multicellular structure, the culture chamber being located below the two or more reservoirs and having fluid communication with each of the two or more reservoirs via separate channels defined by the inserter, A scaffold configured to support organoid formation within the culture chamber, wherein the scaffold is a matrix in which the cells of the multicellular structure are embedded, and A device equipped with the following features.

2. The device according to claim 1, wherein the scaffolding is provided by a module, the module being located in or configured to be located in a slot defined by the inserter.

3. The device according to any one of claims 1 to 2, wherein the shell includes a division having an upper region defining a receptive space and a lower region forming a receptacle, the inserter is configured to be received within the receptive space, and the receptacle is configured to cooperate with the inserter to form the culture chamber.

4. The device according to any one of claims 1 to 3, wherein the inserter forms a slot, defines an opening at the bottom end of the slot, and further comprises a module, the module being configured to be located or positioned vertically above the opening within the slot, and the module being an electrical / magnetic module.

5. A system for culturing multicellular structures, wherein the system is A container comprising a culture chamber for containing the multicellular structure, and two or more reservoirs and slots, each having fluid communication with the culture chamber, Two or more modules, each having different functions, and configured to be interchangeably installed in the slot, A scaffold configured to support organoid formation within the culture chamber, wherein the scaffold is a matrix in which the cells of the multicellular structure are embedded, and A system equipped with these features.

6. The system according to claim 5, wherein the container defines an opening at the bottom end of the slot that communicates with the culture chamber.

7. The system according to any one of claims 5 to 6, wherein the two or more modules include a first module having a permeable membrane.

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