Culture systems, methods, and apparatus for multicellular structures

Abstract

Culture systems, methods, and apparatus for multicellular structures such as organoids. An example system includes a container, an electric / magnetic module, and a control circuit. The container can include a culture chamber to house a multicellular structure. The electric / magnetic module can be configured to be positioned in the container at a location in or adjacent to the culture chamber. The control circuit can be configured to wirelessly power and / or wirelessly operate the electric / magnetic module.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 976,151, filed February 13, 2020, the contents of which are incorporated by reference in their entirety. BACKGROUND

[0003] Organoids (“mini-organs”) are three-dimensional clusters of different types of cells generated in vitro and share certain similarities with organs, such as exhibiting actual tissue architecture of organ-specific tissues. A cell cluster can be generated by seeding a substrate with a small number of stem cells. Then, the stem cells proliferate, differentiate, and self-organize within the substrate while using the substrate as a scaffold. With this approach, organoids similar to tissues such as brain, heart, intestine, kidney, liver, and stomach have been generated so far. These promising results suggest that organoid culture has the potential to provide new insights into organ development and function, and has the potential to reproduce disease models that allow in vitro drug screening. Organoids can revolutionize the way drugs are discovered and personalized.

[0004] Despite the increasing importance of organoids, there are still challenges in effective culture of organoids. New systems, methods, and devices are needed to culture organoids and other multicellular structures. SUMMARY

[0005] The present disclosure provides culture systems, methods, and devices of multicellular structures such as organoids. An exemplary system includes a container, an electric / magnetic module, and a control circuit. The container can include a culture chamber to house a multicellular structure. The electric / magnetic module can be configured to be located in the container and at a position in or adjacent to the culture chamber. The control circuit can be configured to wirelessly power and / or wirelessly operate the electric / magnetic module. BRIEF DESCRIPTION OF DRAWINGS

[0006] FIG. 1 is a block diagram of an exemplary culture system that forms, grows, feeds, differentiates, stimulates, senses, tests, and / or images a multicellular structure, where the culture system includes: a container array that houses the multicellular structure in a respective culture container; at least one electric / magnetic module that is located in at least one culture container of the container array; and a control circuit that powers and / or operates the electric / magnetic module through wireless transmission between the electric / magnetic module and the control circuit.

[0007] FIG. 1A is FIG. 1 is a schematic diagram of an exemplary culture container of the container array of

[0008] FIG. 2 is FIG. 1exploded schematic top view of an embodiment of a container array of the

[0009] FIG. 3 is FIG. 1 exploded schematic top view of another embodiment of a container array of the

[0010] FIG. 4 is FIG. 1A schematic view of a culture container and different functional module groups used in the culture container of the

[0011] FIG. 5 is FIG. 1 block diagram of an exemplary embodiment of a culture container and control circuitry of a culture system of the

[0012] FIG. 6 flowchart of exemplary steps that can be performed in a method of culturing multicellular structures

[0013] FIG. 7 flowchart of exemplary steps that can be performed in a method of culturing multicellular structures using a culture container comprising a housing and an insert

[0014] FIG. 8 is a schematic view of an exemplary electrode module used in a container of the FIG. 1 or a method of the FIG. 6 and a schematic view of a power antenna and a communication antenna of a control circuitry configured to power and operate the electrode module.

[0015] FIG. 9 is a schematic view of an exemplary sensor module used in a container of the FIG. 1 or a method of the FIG. 6 and a schematic view of an antenna of a control circuitry configured to power and operate the sensor module.

[0016] FIG. 10 is a schematic view of an exemplary pump module used in a culture system of the FIG. 1 or a method of the FIG. 6 and a schematic view of a power antenna of a control circuitry configured to power and operate the pump module.

[0017] FIG. 11 is a partial cross-sectional part schematic view of selected aspects of an embodiment of a system of the FIG. 1 comprising two replicas of a pump module of the FIG. 10 which are accommodated in a culture chamber of a culture container and are located at positions adjacent to the organoids.

[0018] FIG. 12 is a partial cross-sectional part schematic view of selected aspects of an embodiment of a system of the FIG. 1partial cross-sectional part view of selected aspects of an embodiment of a system comprising a control circuit, FIG. 11 a culture vessel, and a pair of magnetic modules housed in the culture chamber of the culture vessel, wherein the organoids are located between the magnetic modules.

[0019] FIG. 13 is FIG. 12 another view of an embodiment of the system wherein the coils of the control circuit are energized to drive the magnetic modules to move towards each other in order to mechanically stimulate the organoids.

[0020] FIG. 14 is FIG. 12 a schematic side view of an embodiment of the system, the system variant being configured to culture and mechanically stimulate a group of organoids housed in a row of culture vessels, wherein the culture vessels are omitted and the coils of the control circuit are not energized.

[0021] FIG. 15 is FIG. 14 another schematic side view of a variant embodiment of the system, wherein the coils are energized to drive each pair of magnetic modules to move linearly along the row of culture vessels.

[0022] FIG. 16 is FIG. 14 a schematic top view of a variant embodiment of the system having differently configured energizable coils to drive the magnetic modules to move linearly along the same line.

[0023] FIG. 17 is a schematic view of an exemplary light module for use in a culture vessel of the present disclosure.

[0024] FIG. 18 is a schematic view of an exemplary scaffold module comprising a scaffold attached to the body of the module and configured to support formation and growth of organoids.

[0025] FIG. 19 is a cross-sectional view of an exemplary permeable interface module for use in a culture system of FIG. 1 or a method of FIG. 6 .

[0026] FIG. 20 is a cross-sectional view of a permeable interface module of FIG. 19 operably positioned in a trough portion of a culture vessel housing organoids.

[0027] FIG. 21 is a cross-sectional view of a permeable interface module of FIG. 1 or a method of FIG. 6An isometric view of the frame of an exemplary culture device used in the method, wherein the culture device includes a container assembly forming a row of culture containers, the row of culture containers including a corresponding row of culture chambers, each culture chamber accommodating a pair of magnetic modules, and each culture chamber being positioned adjacent to a corresponding slot accommodating another module that is structurally and functionally different from the magnetic modules.

[0028] FIG. 22 yes FIG. 21 The cultivation equipment is from FIG. 21 The frame was removed and an isometric image was obtained after operatively aligning one of the culture containers of the container assembly with the light source and objective of the imaging system.

[0029] FIG. 23 yes FIG. 21 An isometric exploded view of a culture apparatus, which includes... FIG. 21 or FIG. 22 The cover is not shown in the image.

[0030] FIG. 24 yes FIG. 21 A separate side view of the culture equipment.

[0031] FIG. 25 yes FIG. 21 A separate top view of the culture equipment.

[0032] FIG. 26 It is roughly along FIG. 25 The line 26-26 was cut off FIG. 21 A partial cross-sectional view of the culture equipment.

[0033] FIG. 27 yes FIG. 21 A separate side view of the housing of the culture equipment.

[0034] FIG. 28 yes FIG. 27 A top view of the casing.

[0035] FIG. 29 It is roughly along FIG. 28 The line 29-29 is taken from one of the four sections of the shell. FIG. 28 A cross-sectional view of the shell.

[0036] FIG. 30 It is roughly along FIG. 28 The line is cut at 30-30. FIG. 28 Another cross-sectional view of the shell.

[0037] FIG. 31 yes FIG. 21 A separate side view of the insertion section of the culture device.

[0038] FIG. 32 yesFIG. 31 end view of the insert portion of FIG. 1.

[0039] FIG. 33 FIG. 31 top view of the insert portion of FIG. 1.

[0040] FIG. 34 FIG. 33 cross-sectional view of the insert portion of FIG. 1 taken generally along line 34-34 of FIG. 1. FIG. 31

[0041] FIG. 35 FIG. 21 exploded view of a lid assembly for a container array including a row of container assemblies held by a frame of FIG. 1, wherein the lid assembly includes a series of lids each of which is substantially identical to the lid of FIG. 1. FIG. 23

[0042] FIG. 36 FIG. 31 variant of the insert portion of FIG. 1 including a gasket to form a fluid seal with the housing of FIG. 1. FIG. 27

[0043] FIG. 37 FIG. 36 cross-sectional view of the insert portion of FIG. 1 taken generally along line 37-37 of FIG. 1. FIG. 36

[0044] FIG. 38 top view of an exemplary troughless insert portion placed in the housing of FIG. 1 to form a culture chamber in cooperation with the housing. FIG. 27

[0045] FIG. 39 top view of an exemplary four reservoir insert portion placed in the housing of FIG. 1 to form a culture chamber in cooperation with the housing. FIG. 27

[0046] FIG. 40 top view of an exemplary troughless, four reservoir insert portion placed in the housing of FIG. 1 to form a culture chamber in cooperation with the housing. FIG. 27 DETAILED DESCRIPTION

[0047] ​​​​​​​​​​​​Culturing of large organoids (e.g., up to about 4 mm) is a labor-intensive complex task that can easily take months before the organoids can be harvested or used (e.g., screened). During the culturing, the organoids go through different stages of the culturing protocol as they grow, differentiate, and develop. Currently, most of these stages require manual interaction for feeding, monitoring, handling, etc. Because the culturing vessels that house the organoids can not be suitable for monitoring the status of the developing organoids, it is often necessary to change laboratory equipment. Culturing protocols that rely on manual interaction are costly and prone to handling errors. Furthermore, to culture large organoids that produce specific and interacting cell types, it can be necessary not only to utilize appropriate compounds to treat the cells, but also to expose the organoids to an appropriate physical environment for the type of organoid being cultured. For example, cardiomyocytes and neurons require stimulation by electrical pulses, while muscle and bone organoids rely on the application of alternating mechanical strain. Complex instruments that rely on tubing and wiring can be utilized to culture the organoids. However, such instruments are bulky and non-standardized, and are not easily scalable for culturing many organoids at the same time.

[0048] The present disclosure addresses, in various aspects, the lack of automation, the non-standardization of instruments, the presence of tubing and wiring, and the non- scalability of current performed organoid culturing. More specifically, the containers, modules, and control circuitry provided by the present disclosure enable automation, improved standardization, avoidance of tubing and wiring, and allow scalability.

[0049] The present disclosure provides systems and methods of culturing multicellular structures, such as organoids. The system can include a container including a culturing (chamber) to house the multicellular structure. An electro / magnetic module can be configured to be positioned in the container, and at a location in or adjacent to the culturing chamber. A control circuitry can be configured to power and / or operate the electro / magnetic module via wireless transmission. In the method, the multicellular structure can be housed in the culturing chamber of the container. The electro / magnetic module can be positioned in the container, and at a location in or adjacent to the culturing chamber. The electro / magnetic module can be powered / operated using the control circuitry via wireless transmission of power, force, and / or signals (e.g., data). The electro / magnetic module can include, for example, a magnet that can be moved in the container by a magnetic field generated by the control circuitry, such that the module pumps fluid or mechanically stimulates the multicellular structure housed in the culturing chamber. In other examples, the electro / magnetic module can include electrodes that stimulate the multicellular structure, sensors that sense properties of the multicellular structure and / or the culture medium in the container, light sources that illuminate at least a portion of the multicellular structure, etc.

[0050] The ability to wirelessly power / operate the electrical / magnetic modules in the system and method is important because it eliminates the need for wiring or other electrical conductors extending from the control circuit to the vessel. As a result, the vessel becomes more portable, has less restrictions on positioning relative to the control circuit, and is more easily separable from the control circuit (e.g., to perform an imaging procedure). Moreover, this wireless approach can improve fidelity and stability of multi-cellular structure experiments because it allows the electrical / magnetic modules to be positioned in close proximity to or in contact with the multi-cellular structure under analysis. The system and method also facilitate automation of complex culture of multi-cellular structures by providing the necessary physical environment, and enable multi-channel in-situ monitoring of multi-cellular structures. The system and method enable simultaneous stimulation, maintenance, and monitoring of a large number of organoids in an automated manner.

[0051] The vessel of the present disclosure can be customized as desired by introducing one or more active / passive modules into one or more compartments of the vessel. The selection of one or more functional modules and one or more compartments to house the one or more modules allows for functional adjustment of the vessel during manufacturing and / or by the user. For example, the selection of one or more modules and one or more compartments can enable culture of a particular type of organoid, execution of a particular culture protocol or protocol phase, creation of one or more desired test conditions, and / or on-board (in the vessel) sensing, measurement, and / or monitoring of one or more desired parameters, among others. Thus, the base structure of the vessel can be standardized while the functionality of the vessel can be modified as desired by introducing different modules to suit the particular needs of various users. Moreover, introduction of one or more modules into the vessel allows the modules to be in close proximity to or in contact with the multi-cellular structure, such that the modules have more direct interaction with the multi-cellular structure. Furthermore, introduction of one or more modules into the vessel can not result in any increase in footprint size. Thus, the vessel modified with one or more modules can remain compact, which allows more vessel replicates to fit into the footprint of a standard microplate, which in turn allows more multi-cellular structures to be cultured simultaneously in an incubator.

[0052] Culture apparatus and methods for multicellular structures such as organoids are provided. The apparatus can include a housing having an open top. The apparatus can also include an insert having two or more reservoirs. The insert can be configured to be received in the housing via the open top such that the housing and the insert cooperate to form a culture chamber for a multicellular structure. The culture chamber can be located below the two or more reservoirs and in fluid communication with each of the two or more reservoirs via respective channels defined by the insert. In the methods, an insert including two or more reservoirs can be placed in a housing so as to use the insert and the housing to cooperate to form a culture chamber. The culture chamber can be located below and in fluid communication with each of the two or more reservoirs. A multicellular structure can be cultured in the culture chamber.

[0053] The apparatus and methods described in the preceding paragraphs can provide various advantages for the culture of multicellular structures such as organoids, including any combination of the following. The insert can be selected from a group of inserts having different characteristics from one another, such as different reservoir, channel, and / or trough configurations. Thus, the same housing can be assembled with different types of inserts to customize the structure of the final container according to the specific needs of the user. Furthermore, the insert can position the reservoirs (and optional troughs) vertically above the culture chamber so that gravity can drive fluid flow into and / or out of the culture chamber. Moreover, the housing can define a row of sections, each of which can receive an insert to form a respective culture chamber. Thus, the housing and two or more inserts can be assembled to form a container assembly having a row of culture chambers. Furthermore, two or more container assemblies can be held by a frame having a footprint corresponding to the footprint of a standard microplate to enable formation of a compact array of containers. Each container assembly can be individually removed from the frame for separate handling from the other container assemblies of the array.

[0054] The container can be provided with a plurality of reservoirs in fluid communication with a chamber (culture chamber) via channels that can be formed in one or more shared walls between the reservoirs and the chamber. Such a configuration can be described as a standard feeding interface. In some embodiments, 3D printing provides the connection of the standard feeding interface within the container to any suitable printed structure to enable growth of different types of organoids.

[0055] The matrix can provide a temporary scaffold for the appropriate type of cells as they develop into organoids. The cells can self-organize and produce their own extracellular matrix that can replace some or all of the scaffold. The same is true for internal feeding: the container can provide a general interface that can be optionally modified by 3D printing, and the cells can be organized to use the modified interface in an optimal way.

[0056] In some embodiments, a scaffold (with or without cells) can be disposed in the containment portion of the container body, and optionally, upon inverting the container, the containment portion can be formed into a culture chamber using a sealing member. Once these processes are complete, the container can be flipped right-side up (flipped to the organoid culture orientation of the container), and at least one reservoir overlying the culture chamber can be filled with a feeding solution. If no cells are already present within the scaffold, suitable cells can be placed in the feeding solution, and the suitable cells introduced into the scaffold from the reservoir overlying the culture chamber along with the feeding solution.

[0057] Prior to commencing a particular feeding regimen, an initial incubation time can be required to form an organoid. The feeding regimen can include loading and removing culture media from the reservoirs with suitable culture media (media) according to a predetermined schedule and / or based on the developmental stage or condition of the organoid. The feeding regimen can depend on the shape of the scaffold and the type of organoid to be formed.

[0058] The container enables light sheet 3D imaging. The culture chamber of the container can have two, three, or more optical windows, and light can be propagated into and / or out of the culture chamber via each optical window. For example, the container can have a bottom window and one or more lateral windows, each of which can be planar. In some embodiments, the container can have a pair of lateral optical windows arranged opposite one another.

[0059] The present disclosure enables the production of large functional organoids. The average or maximum diameter of a large organoid can be greater than about 0.1, 0.2, 0.5, 1, or 2 millimeters, among others. Working with large organoids remains challenging, and researchers face two main limitations. First, each type of organoid can require different culture conditions, such as a particular hydrogel as a scaffold, or even mechanical stimulation like shear forces of media flow. Second, microscopy of large organoids can be very challenging. The most advanced methods currently are still thin sectioning of organoid material, staining, and image acquisition of fixed samples using confocal scanning microscopy or even slide readers.

[0060] The present disclosure provides systems, methods, and apparatus for improved organoid culture. By using a combination of 3D printing (scaffolds and / or cells) with gravity flow exchange of media, the user can generate an optimized unique 3D environment for each type of organoid. A variety of different organoid types can be grown. Feeding and waste removal can be addressed through fluid communication between a culture chamber and a storage portion of a vessel. Integrating optical windows with each vessel, at least one optical window for the entry of excitation light and another optical window for the exit of emitted light, allows for live cell monitoring of the organoids through light sheet microscopy. Alternatively or additionally, the organoids can be imaged through classical widefield microscopy via one or more optical windows. Thus, the vessels disclosed herein are capable of performing live cell microscopy of developing organoids and / or developed organoids. High capacity and / or high throughput microscopy of organoids can be performed.

[0061] Other aspects of the present disclosure are described in the following sections: (I) Definitions, (II) Culture System and Method Overview, (III) Electrical / Magnetic Modules, (IV) Passive Modules, (V) Vessel Assembly, and (VI) Selected Aspects.

[0062] I. Definitions

[0063] Technical terms used in the present disclosure have meanings generally recognized by those skilled in the art. However, the following terms can also be defined as follows.

[0064] Cells —The basic structural, functional, and biological unit of an organism. Cells can be eukaryotic or prokaryotic. Exemplary cells include stem cells, differentiated cells, established cells (e.g., cell lines), primary cells, cells of a tissue sample, transfected cells, cells from a clinical sample (e.g., a blood sample, a fluid aspirate, a tissue explant, etc.), cells that form a complete organism, etc.

[0065] Any suitable cells can be introduced into the culture chamber (or into a containment portion that will form part of the culture chamber). The introduced cells can include stem cells (e.g., pluripotent stem cells), support cells, etc. The cells can be deposited in the culture chamber or containment portion and / or in a scaffold located or to be located in the culture chamber or containment portion by any suitable technique, including pipetting, bio-ink droplet printing, microcontact printing, photolithography, dip-pen nanolithography, etc.

[0066] Cell Culture —Promoting the survival, health, growth, proliferation, differentiation, and / or self-organization of living cells, such as cells of a multicellular structure, in an artificial environment.

[0067] Culture Chamber—accommodates a multi-cellular structure and has walls on substantially all sides of the enclosure. At least one wall can define one or more openings to allow communication with and / or access to the enclosure.

[0068] Culture Medium —aqueous compositions for cell culture. The composition can be a liquid or semi-solid. The composition can include a carbon source (e.g., glucose), inorganic salts, vitamins, and growth regulators, etc. As used herein, the term “media” refers to at least one medium and can refer, for example, to a separate volume of media, a first medium and a second medium of different composition, a medium of substantially the same composition in contact with a different / separate cell culture, or a combination / mixture of previously separate volumes of the same medium.

[0069] Culture Vessel —culture apparatus for multi-cellular structures. Culture vessels (interchangeably referred to as vessels) can include a culture chamber and one or more reservoirs in fluid communication with the culture chamber. A vessel assembly or vessel array is a collection of culture vessels for culturing multi-cellular structures in one-, two-, or three-dimensional arrangements. The culture vessels disclosed herein can be single-use apparatus (consumables) or can be reusable.

[0070] Exemplary —illustrative or used as an example. Similarly, the term “example” refers to by way of illustration. Neither of these terms implies desirability or superiority.

[0071] In —“in” or “internal” when describing the location / orientation of an object relative to a given structure means that the object is present at least predominantly (more than 50% of the object’s volume) or entirely within the given structure. In the same context, “external” means that the object is present at least predominantly (more than 50% of the object’s volume) or entirely outside of the given structure.

[0072] Light —optical radiation includes ultraviolet radiation, visible radiation (i.e., visible light), and / or infrared radiation.

[0073] Module— structurally and functionally discrete units configured to be housed in the culture vessel. The modules can be insertable into and / or removable from the culture vessel while the culture vessel remains intact or only when the culture vessel is disassembled, or the modules can be configured to be non-removable from the culture vessel. The modules can be active modules, also referred to as electric / magnetic modules, which are modules that operate using electricity and / or magnetism, and optionally powered / operated through wireless transmission between a control circuit and the module. Alternatively, the modules can be passive modules, which are modules that do not use electricity and / or magnetism to achieve one or more intended purposes of the passive module. Electric / magnetic modules include magnetic modules that include permanent magnets but no electrical / electronic devices, electric modules that include electrical / electronic devices but no permanent magnets, and modules that include both permanent magnets and electrical / electronic devices. Magnetic modules can require an externally generated and optionally time-varying magnetic field to operate, such as to drive the magnetic module and / or movement of the magnets of the magnetic module.

[0074] The modules can have any suitable shape and size. The modules, and in particular the housing or body of the modules, can be, for example, cuboid (e.g., cubical), cylindrical, conical, etc. The cross-sectional shape of the modules can correspond to the shape of the troughs or other compartments of the vessel, so that the modules fit into the troughs or other compartments. To provide flexibility and interchangeability, it can be advantageous to have all modules configured to be placed in a given compartment of the vessel (e.g., a trough of the vessel) have the same standard shape and size, or to have all modules have the same standard size and shape regardless of the compartment purpose. 4 x 4 x 4 mm 3 are exemplary dimensions of active and passive modules that correspond to the dimensions of large organoids that can be generated in the culture chamber of the vessel.

[0075] Multicellular Structure — a three-dimensional arrangement of biological cells connected to each other. The multicellular structure can be a organized multicellular structure, which is a multicellular structure composed of different cell types that are non-randomly arranged relative to each other. Exemplary multicellular structures include organoids, organisms (at any stage of development), tissue explants, tumors, etc.

[0076] Near Field Communication (NFC) — wireless communication between electronic devices using near-field radiation and inductive or capacitive coupling. Near-field communication can be performed when the electronic devices are within a distance of 50, 20, 10, or 5 centimeters of each other.

[0077] Near Field Radiation — electromagnetic radiation, typically radio waves (e.g., microwaves), located within 10, 5, or 2 wavelengths from the source of the radiation, such as within 50, 20, 10, or 5 centimeters from the source of the radiation.

[0078] Organoid Three-dimensional aggregates of different types of cells produced in vitro and having some similarities to organs, such as exhibiting actual tissue architecture of organ-specific tissues. Cell aggregates can be produced by seeding scaffolds (i.e., matrices) with small numbers of stem cells. The stem cells then proliferate, differentiate, and self-organize within the scaffold.

[0079] Receptacle Optionally, the housing has an open side, such as an open top side, an open bottom side, or an open lateral side. By at least partially covering or closing the side of the housing, the housing can be converted into a culture chamber.

[0080] Scaffold An extracellular support framework for culturing a multicellular structure. The scaffold is typically a matrix in which or onto which cells of the multicellular structure are or will be embedded. The scaffold can be provided by one or more hydrogels. Each hydrogel can include one or more thermoplastic structural components, such as Matrigel, alginate, nanofibrillar cellulose, collagen, fibrin, and / or polyethylene glycol, that cooperate in a temperature-dependent manner to form the matrix.

[0081] In some embodiments, two or more different hydrogels / matrices can be disposed in the culture chamber of the container. The hydrogels / matrices can differ with respect to any suitable parameter, such as melting temperature, resistance to enzymatic degradation, solubility, cell attraction, and / or cell repulsion properties.

[0082] Each hydrogel / matrix can include any suitable components. Exemplary components include one or more polysaccharides (e.g., glycosaminoglycans (such as chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, hyaluronan, keratan sulfate, etc. GAGs), proteoglycans (e.g., GAGs linked to a core protein (such as via a serine of the core protein) to form aggrecan, agrin, brevican, collagen type XVIII, decorin, neurocan, perlecan, slitam, versican, etc.), fibrous proteins (e.g., collagen, elastin, fibronectin, laminin, etc.), etc.). Protease recognition sites (e.g., for matrix metalloproteinases (MMPs)) can be incorporated into the hydrogel / matrix to allow for degradation / remodeling by cells. The frequency of such sites and the order of each site can be selected to allow for an appropriate amount of degradation / remodeling.

[0083] One or more growth factors can be included in the matrix when the matrix is formed, or the one or more growth factors can be introduced into the culture medium after the matrix is formed. Exemplary growth factors that can be suitable include angiogenin, bone morphogenetic protein (BMP), ciliary neurotrophic factor, colony stimulating factor, epinephrine, epidermal growth factor, erythropoietin, fibroblast growth factor, glial derived neurotrophic factor, hepatocyte growth factor, insulin, insulin-like growth factor, interleukin, leukemia inhibitory factor, keratinocyte growth factor, neuregulin, neurotrophin, platelet derived growth factor, transforming growth factor, tumor necrosis factor (alpha), vascular endothelial growth factor, and the like.

[0084] II. Culture System and Method Overview

[0085] This section provides an overview of the culture systems and methods of the present disclosure; see FIGS. 1-7 .

[0086] FIG. 1 An exemplary culture system 100 is shown that forms, grows, differentiates, organizes, stimulates, senses, analyzes, and / or images multicellular structures such as organoids. The culture system 100 includes a container array 101 that includes a set of culture containers 102, FIG. 1 Only three of the set of culture containers 102 are explicitly identified in the figure. Each culture container 102 is configured to house a respective multicellular structure. The container array 101 can 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. The container array 101 and / or each of two or more discrete container assemblies of the container array 101 can have a footprint corresponding to the length and / or width of a standard microplate to facilitate mechanical and fluidic processing with robotic systems designed to manipulate standard microplates. The number of culture containers 102 in the container array 101 can be at least three in a first dimension and one or more (e.g., at least two, three, or more) in a second orthogonal dimension.

[0087] Each culture container 102 of the container array 101 can include two or more distinct compartments that can or can not be in fluid communication with each other and can or can not share one or more walls with each other (see FIG. 1A ). The culture container 102 has a culture chamber 103 to house a multicellular structure 104 such as an organoid. At least one reservoir 105 of the container 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 respective connection channel 106. The container 102 can also have at least one sink 107 that can be in communication with the culture chamber 103 via a hole 108.

[0088] Each compartment of the culture vessel 102 can have any suitable size and shape. The culture chamber 103 can have a volume of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, or 1 ml. The size of the culture chamber can be designed to accommodate a multicellular structure such as an organoid of any suitable 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 capacity that is greater than the culture chamber 103, such as at least 2, 5, or 10 times the volume of the culture chamber, and / or at least 0.5, 1, 2, 4, or 6 ml, etc. The trough 107 can have a capacity that is greater than, less than, or equal to the culture chamber 103 or one or more reservoirs 105, and / or can have the same height as each reservoir 105. The horizontal cross-section of each compartment of the vessel can be rectangular, circular, oval, etc. It can be advantageous for each compartment of the vessel to have a rectangular cross-section, as this shape makes very efficient use of the available space and allows the culture vessel 102 to be arranged in close proximity to one another in the vessel array 101.

[0089] The culture vessel 102 can be formed from any suitable material or materials by any suitable process. In exemplary embodiments, the culture vessel can be constructed from at least one polymer, which can include a transparent polymer. The culture vessel 102 can be formed integrally as a single piece, or from at least a pair of discrete components (such as a housing and an insert, or a main body and a sealing member) that fit together and / or are attached to one another to form the culture chamber 103. Thus, the culture chamber 103, reservoirs 105, and / or trough 107 can have fixed positions relative to one another and / or can be non-removably / securely attached to one another in the culture vessel 102.

[0090] One or more reservoirs 105 of the container 102 can hold any suitable substance for supply to the culture chamber 103. Exemplary substances include nutrients, effectors, and reagents, among others. Suitable nutrients include any substance that contributes to cell health and proliferation and thus to the growth and development of multicellular structures such as organoids within the culture chamber 103. Exemplary nutrients can include sugars (e.g., glucose), amino acids, proteins, nucleotides, vitamins, minerals, fatty acids, and the like. Effectors include any molecule (such as an inducer or repressor) that activates, controls, or inactivates a process or action such as differentiation, protein synthesis, migration, and the like. Exemplary effectors include anti-cancer compounds, growth factors, differentiation factors, oligonucleotides, mRNAs, and the like. Reagents include any compound that facilitates analysis of multicellular structures such as organoids. Exemplary reagents include markers, fixatives, scavengers, and the like. Markers can include dyes (e.g., visible colorants and / or photoluminescent dyes). Photoluminescent dyes are any substance that emits light in response to irradiation by electromagnetic radiation such as excitation light.

[0091] Each reservoir 105 can have an open top to facilitate introduction and removal of fluids using a fluid transfer device (e.g., a pipette). A lid can be provided that rests on the culture container 102 to cover the open top of each reservoir 105 during incubation in the incubator. The lid can have a flange configured to overlap the upper region of each reservoir in the vertical direction when covering one or more reservoirs and to limit lateral movement of the lid, while optionally not creating a tight fit. In some embodiments, the lid can be a closure that forms a fluid seal at the top of one or more reservoirs 105.

[0092] The container 102 can house at least one module 109 in a compartment of one or more containers. FIG. 1A Modules 109 are shown positioned in the culture chamber 103, with possible alternative positions of the same modules shown in dashed outline. More specifically, each module 109 can be positioned in the culture chamber 103, a reservoir 105, or a slot 107, among others. Thus, each module 109 can be positioned in or adjacent to the culture chamber 103. Each module 109 can be housed in the culture container 102, or can be present in a set of functionally distinct modules 109 that are positioned outside the culture container 102 and configured to be selectively placed in the culture container 102 by a user. As described below, each module 109 can independently be an electric / magnetic active module (e.g., an electrode module, an actuator module, a sensor module, a pump module, and / or an illumination (light emitting) module, among others) or a passive module (e.g., a permeable interface module, a scaffold module, or a dummy module).

[0093] The culture system 100 can also include an incubator 110 to house the container array 101 (see FIG. 1). The incubator temperature can be controlled to a suitable culture temperature for the multicellular structures 104, such as at least 25°C, 30°C, or 35°C, etc. (see also FIG. 1 The humidity and / or atmosphere inside the incubator 110 can also be controlled to promote the growth and development of the multicellular structures 104.

[0094] The culture system 100 can also include a fluid delivery system 111, a detection system 112, and a conveyor 113, each of which can be located inside or outside the incubator 110. The fluid delivery system 111 is configured to add and / or remove liquid from each culture vessel 102 of the vessel array 101, such as adding and / or removing liquid from each reservoir 105 of each culture vessel 102. The fluid delivery system can thus include one or more pipettes, fluid supplies, and / or waste receptacles. The detection system 112 is configured to collect data related to the multicellular structures housed by the vessel array 101, such as by optical detection. For example, the detection system 112 can include a light source 114 that illuminates at least a portion of the multicellular structures in each culture vessel 102, and an image sensor 115 that captures an image of at least a portion of the multicellular structures. The conveyor 113 can be configured to move components of the culture system 100 relative to one another. For example, the conveyor 113 can be configured to move the vessel array 101 as a unit or only a portion of the vessel array 101 within the culture system 100. For example, the conveyor 113 can be configured to move the vessel array 101 or culture vessels 102 of the vessel array 101 into and / or out of the incubator 110, to and / or from the fluid delivery system 111, and / or to and / or from the detection system 112. Alternatively or additionally, the conveyor 113 can be configured to remove and replace one or more covers (if present) covering the vessel array 101 when accessing the reservoirs 105 and / or the well portions 107.

[0095] A control circuit 116 of the culture system 100 powers and / or operates any suitable equipment of the culture system. For example, the control circuit 116 can control each of the fluid delivery system 111, the detection system 112, and / or the conveyor 113 via wired or wireless communication, which can be one-way or two-way communication. As shown by the dashed arrows at 117, each module 109 held by the vessel array 101 can also be controlled wirelessly by the control circuit 116. Such wireless control is advantageous because it simplifies the construction of the vessel array 101, allows functional customization of the vessel array by introducing appropriate modules as needed, and eliminates the need for wiring or electrical conductors extending into the culture vessels and providing a pathway for microbial contamination.

[0096] FIG. 2 An exemplary container array 201 of the culture system 100 of FIG. 1 is shown. The container array includes a plurality of container strips 218 (interchangeably referred to as container assemblies), each container strip 218 including a row of culture containers 202 attached to one another, e.g., via a common (shared) housing. The container strips can have any suitable number of culture containers 202, such as at least two, three, four, or more. Each container strip 218 is housed and held by a frame 219, which optionally forms a row of container strips 218. For example, each container strip 218 is placed as a unit in a corresponding housing site of the frame 219, either during manufacturing or by a user. In the depicted embodiment, the frame 219 has a row of eight housing sites to house a corresponding number of container strips 218, but in other embodiments, the frame can be configured to house at least two, three, or more container strips 218 in a corresponding number of housing sites. Each container strip 218 can be removed individually from the frame 219 to allow the container strip (and / or the contents of the container strip) to be manipulated, processed, and / or analyzed separately from the other container strips 218 of the container array 201.

[0097] FIG. 3 Another exemplary container array 301 of the culture system 100 of FIG. 1 is shown. The container array includes a plurality of separate, individual culture containers 302 housed and held by a frame 319. For example, each culture container 302 is placed in a corresponding opening of the frame 319, either during manufacturing or by a user. The culture containers can or can not be removable from the frame.

[0098] FIG. 4 An exemplary container array 201 of the culture system 100 of FIG. 1A is shown. The container array includes a plurality of container strips 218 (interchangeably referred to as container assemblies), each container strip 218 including a row of culture containers 202 attached to one another, e.g., via a common (shared) housing. The container strips can have any suitable number of culture containers 202, such as at least two, three, four, or more. Each container strip 218 is housed and held by a frame 219, which optionally forms a row of container strips 218. For example, each container strip 218 is placed as a unit in a corresponding housing site of the frame 219, either during manufacturing or by a user. In the depicted embodiment, the frame 219 has a row of eight housing sites to house a corresponding number of container strips 218, but in other embodiments, the frame can be configured to house at least two, three, or more container strips 218 in a corresponding number of housing sites. Each container strip 218 can be removed individually from the frame 219 to allow the container strip (and / or the contents of the container strip) to be manipulated, processed, and / or analyzed separately from the other container strips 218 of the container array 201.

[0099] FIG. 5 An exemplary container array 201 of the culture system 100 of FIG. 1An exemplary culture vessel 502 and exemplary control circuit 516 of a culture system 100. The culture vessel 502 is shown in schematic side view and includes a culture chamber 503 that houses a multicellular structure 504 (i.e., an organoid 521) and an associated scaffold 522 to support formation and / or growth of the organoid. In FIG. 5 The scaffold 522 is attached to a bottom wall of the culture chamber 503, but in other embodiments, the scaffold 522 can be attached to any suitable one or more lateral walls or top wall of the culture chamber. At least one pair of reservoirs 505a and 505b are positioned above the culture chamber 503 and communicate with the culture chamber via respective channels 506a and 506b. Each reservoir can be "vertically above" the culture chamber, meaning a vertical line extends through the reservoir and the culture chamber. Each of the reservoirs 505a and 505b holds a respective culture medium 523a and 523b, which can have the same or different composition from one another. The culture chamber 503 also holds a culture medium, which can be at least partially supplied by one or both of the reservoirs 505a and 505b via one or both of the channels 506a and 506b.

[0100] The culture vessel 502 optionally defines a slot 507 positioned above the culture chamber 503. The slot 507 can be described as an access slot, as the slot can communicate with the culture chamber 503 via an aperture 508 at a bottom end of the slot. The aperture 508 can (or can not) have a diameter that is greater than each of the channels 506a and 506b, such as at least 50% or 100% greater. The slot 507 is configured to receive a slot module 509a, which can be placed in the slot 507 during manufacturing or by a user. In some cases, the user can select the slot module 509a from a set of functionally different slot modules to place in the slot 507 (see, e.g., FIG. 4 The ability to interchange slot modules of different functionality enables the culture vessel 502 to accommodate different culture / testing mechanisms for various types of organoids or other multicellular structures. In some cases, a dummy module can be placed in the slot 507 to cover the aperture 508 if a slot module is not used in the slot.

[0101] The slot 507 can have any suitable position relative to the culture chamber 503 and the reservoirs 505a and 505b. As shown, the slot can be centered between the reservoirs, or can have a lateral position relative to the reservoirs.

[0102] The trough portion 507, as well as the storage portions 505a and 505b, can be open at their respective top ends. Such a configuration allows for placement of a trough module 509a in the trough portion 507, and for dispensing of culture media 523a and 523b into one or both storage portions. Accordingly, the container 502 can include a removable lid to cover the open top of the trough portion 507 and / or the storage portions 505a and 505b. Additional aspects of the culture container and lid of the container assembly are described in Section V below.

[0103] The culture (chamber) 503 can house at least one chamber module 509b. The chamber module can be placed in the housing 524, for example, via the top side, bottom side, or lateral side of the housing 524, and the housing 524 can be converted into the culture chamber 503 by at least partially closing the top side, bottom side, or lateral side of the housing. In some cases, if the chamber module 509b is large enough, the chamber module can be left in the culture chamber 503 unless and until the culture chamber is opened and / or disassembled by removing a wall portion of the culture chamber.

[0104] If one or both of the trough module 509a and / or the chamber module 509b is an electric / magnetic module, the control circuit 516 is configured to wirelessly control (as shown at 517) the trough module 509a and / or the chamber module 509b (see Section III). The control circuit can include one or more antennas 525 to transmit / receive power and / or data to each of the modules 509a and 509b (if present) that are electric modules using near-field radiation. One or more coils 526 of the control circuit can be used to generate a magnetic field to drive movement of at least a magnetic portion (if present) of each of the modules 509a and 509b. The control circuit 516 can also include a computer including a processor 527, one or more controllers 528, a memory 529, and / or a user interface 530 (e.g., a display, keyboard, mouse, printer, etc.).

[0105] The control circuit 516 can be configured to control movement and / or operation of one or more electric / magnetic modules housed in each culture container of the culture container array. Accordingly, for each culture container of the array, the control circuit can have at least one respective antenna 525 and / or at least one respective coil 526.

[0106] FIG. 6 Flowchart 630 of exemplary steps 631a-631j can be performed in any suitable order and combination to provide a method of culturing a multicellular structure. The method can be performed using any suitable system, device, cells, and scaffold of the present disclosure.

[0107] At step 631a, a scaffold can be placed in the containment portion or culture chamber of the culture vessel. The scaffold can be provided by forming the scaffold in the containment portion or culture chamber, such as by 3D printing, or by placing a preformed scaffold in the containment portion or culture chamber. In some examples, the scaffold can be attached to a scaffold module that can be placed in the trough portion of the culture vessel, either before or after the culture chamber is formed.

[0108] At step 631b, cells can be introduced into the containment portion or culture chamber of the culture vessel. The cells can include stem cells that are intended to produce organized multicellular structures through differentiation, division, migration, etc. In other cases, the cells can be introduced into the containment portion or culture chamber as preformed multicellular structures (e.g., organisms, tissue explants, tumors, organoids, etc.). The cells can be introduced during step 631a, or can be introduced before or after step 631a.

[0109] At step 631c, one or more modules can be placed in or adjacent to the containment portion or culture chamber. Any combination of modules as disclosed herein can be placed. Any suitable number of one or more modules can be placed during manufacture of the vessel and / or by a user.

[0110] At step 631d, a culture chamber of the culture vessel can be formed. The culture chamber can be formed using the containment portion, which can have an open side, by at least partially closing the open side. For example, the open side can be covered by bonding a sealing member to the containment portion at the open side (e.g., to the open bottom side of the vessel) or by placing an insert portion in a housing that includes the containment portion. In each case, the containment portion and the sealing member or insert portion can be used to cooperatively form the culture chamber.

[0111] At step 631e, a module can be placed in the trough portion of the vessel. The module can be placed before or after step 631d. In some cases, a scaffold module that includes a preformed scaffold can be used to perform steps 631a and 631e together. In some cases, a module that includes a battery can be used to perform steps 631b and 631e together.

[0112] At step 631f, culture medium can be added to one or more storage portions of the culture vessel. Once added, the culture medium can optionally flow from one of the storage portions into the culture chamber driven by gravity. Gravity can also drive the flow of culture medium out of the culture chamber and into different storage portions of the culture vessel.

[0113] At step 631g, the container and contents of the container can be incubated. Incubation can be performed at a suitable temperature and in a suitable gas atmosphere, and for any suitable length of time, such as at least 1, 2, 3, 4, or 5 days, or at least 1, 2, or 3 weeks, etc.

[0114] At step 631h, each electrical / magnetic module in the culture container (if present) can be wirelessly controlled by the control circuit. Such control can include transmitting power, force, and / or data to the module to drive movement of the module or at least a portion of the module, and / or to control operation of the module. Step 631g and step 631h can be performed simultaneously.

[0115] At step 631i, the multicellular structure can be formed, grown, stimulated, and / or sensed in the culture chamber. Step 631i can be performed in response to step 631g and / or step 631h.

[0116] At step 631j, a first module housed by the container can be removed and replaced by a second module. The first module can be removed from a slot portion of the container and replaced by a second module in the same slot portion. The first module and the second module can be functionally different from one another. After replacing the first module with the second module, step 631h and step 631i can be repeated.

[0117] FIG. 7 Flowchart 730, which is an example of steps 731ab, 731d, 731f, 731g, and 731i, can be performed in any suitable order and combination, optionally adding one or more steps of flowchart 630 to provide a method of culturing a multicellular structure. The method can be performed using any suitable system, device, cells, and scaffold of the present disclosure. Steps of flowchart 730 that correspond to steps of flowchart 630 have the same letter designations in both flowcharts. FIG. 6

[0118] At step 731ab, a scaffold and / or cells can be placed in a housing of a container. More specifically, the scaffold and / or cells can be disposed in a receptacle formed by a portion of the housing.

[0119] At step 731d, an insert portion of the container is placed in the housing to form a culture chamber. The culture (chamber) can be formed by the receptacle and the housing cooperating. The receptacle can provide a bottom wall and lateral walls of the chamber, and the insert portion to provide a top wall of the culture chamber. Step 731d can be performed before or after the scaffold and / or cells are disposed in the receptacle.

[0120] At step 731f, a culture medium can be added to at least one storage portion above (e.g., vertically above) the culture chamber. The insert portion can provide the at least one storage portion or each storage portion above the culture chamber.​

[0121] At step 731g, the culture vessel and contents of the vessel can be incubated. As described above at step 631g (see FIG. 6 ), incubation can be performed at a suitable temperature and in a suitable gas atmosphere, and for any suitable length of time.

[0122] The method of FIG. 7 and Electrical / Magnetic Module can be performed using an array of culture vessels. Thus, each step of the method can be performed in groups on the array of culture vessels, or individually on the culture vessels, as practical.

[0123] III. FIGS. 8-17

[0124] This section describes exemplary electrical / magnetic modules for use in the culture systems and methods of the present disclosure; see FIG. 8 , each electrical / magnetic module is configured to be wirelessly powered / operated by a control circuit while the module is located in a culture vessel. The electrical / magnetic module utilizes electricity, magnetism, or a combination thereof, that is wirelessly supplied or applied to the module by the control circuit to drive movement of at least a portion of the module relative to the vessel and / or operation of at least one electrical / electronic device of the module. The module can be configured to be housed in any culture vessel of the present disclosure, as well as in any one or more compartments of that any culture vessel, such as a trough portion, one or more storage portions, and / or a culture chamber of the culture vessel.

[0125] FIG. 9 An exemplary electrode module 809 (i.e., an electrical / magnetic embodiment of module 109) is shown, and also a power antenna 825a and a communication antenna 825b of control circuit 816. Antennas 825a and 825b are configured to transmit power to and operate electrode module 809, respectively.

[0126] The electrode module 809 includes a housing 832 that supports an electrode interface 833. The electrode interface can be located on any suitable side of the housing 832, such as the bottom side as shown or a lateral side of the housing 832 (e.g., if the electrode module 809 is housed in a culture chamber), etc. The electrode interface 833 can include any suitable number of electrodes of any suitable shape. For example, the electrode interface can have a pair of plate electrodes 834a that can be configured to contact a surface of a multicellular structure in a culture chamber and / or a pair of needle electrodes 834b that can be configured to penetrate and extend into a multicellular structure. The electrode interface 833 can be used to electrically stimulate and / or externally or internally sense electrical properties of a multicellular structure. Example uses of the electrode module 809 include electrically stimulating (a) cardiomyocytes or neurons for pacing or activation, (b) myoblasts to promote muscle differentiation and three-dimensional growth, or (c) neural tissue (e.g., brain organoids) to promote axon growth and morphological changes to influence networks, etc. Other example uses of the electrode module 809 include sensing electrical activity of any suitable cells / tissue, such as neurons, smooth muscle, cardiomyocytes, or skeletal muscle.

[0127] The housing 832 can house any suitable electronic circuitry to enable wireless power, communication with, and / or control of the electrode module 809. The circuitry can include a power receiver 835 with an antenna to receive power wirelessly transmitted from the power antenna 825a of the control circuitry 816. The circuitry can also include an internal communication antenna 836 to receive and / or transmit signals to / from the external communication antenna 825b of the control circuitry 816 to enable communication between the electrode module 809 and the control circuitry 816. Any suitable communication protocol can be utilized, such as at least one near field communication (NFC) protocol. The electronic circuitry can also include a power storage unit 837 to store power (energy) for the power receiver 835, a controller 838, and a digital-to-analog converter (DAC) and pulse generator 839. The electronic circuitry of any of the modules of Section III can be sealed inside the housing to prevent damage to the electronic circuitry when the module is in contact with a liquid, such as a culture medium, etc.

[0128] FIG. 8 An example sensor module 909 (i.e., an electrical / magnetic embodiment of the module 109) is shown, and the communication antenna 925 of the control circuitry 916 is also shown. The antenna 925 is configured to transmit power to and operate the sensor module 909. Thus, a separate power antenna and power receiver can not be necessary (as compared to FIG. 10

[0129] ​The sensor module 909 includes a housing 932 that supports a sensor interface 940. The sensor interface can be located on any suitable side of the housing 932, such as the bottom side as shown or a lateral side of the housing 932 (e.g., if the electrode module 909 is housed in a culture chamber), etc. The sensor interface 940 can be configured to sense and measure any suitable physical or chemical parameter, such as temperature, motion, electrical parameters (e.g., potential, current, impedance, etc.), electric / magnetic fields (e.g., with a Hall effect sensor), pH, chemical potential, oxygen or carbon dioxide concentration, compounds (e.g., with an electrochemical sensor), etc. The sensor module 909 can be suitable 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.

[0130] The housing 932 can house any suitable electronic circuitry to enable wireless powering, communication with, and / or control of the sensor module 909. The circuitry can include an internal communication antenna 936 to receive and / or transmit signals from and to the external communication antenna 925 of the control circuit 916 to power and operate the sensor module 909. Any suitable communication protocol can be utilized, such as at least one near field communication (NFC) protocol. The electronic circuitry can also include a controller 938, an analog-to-digital converter (ADC) 941, a memory 942, and amplifiers and sensor electronics 943.

[0131] FIG. 8 An exemplary pump module 1009 (i.e., an electrical / magnetic embodiment of the module 109) is shown, and a power antenna 1025 of the control circuit 1016 is also shown. The power antenna 1025 is configured to transmit power to the pump module 1009. A separate communication antenna can or can not be present in the control circuit 1016 (as compared to the module 109). FIG. 11

[0132] The pump module 1009 includes a housing 1032 that houses a pump 1044 to drive fluid flow. The pump 1044 is in fluid communication with a pair of nozzles 1045a and 1045b located at a periphery of the pump module 1009. Operation of the pump 1044 draws fluid into the nozzle 1045a (serving as an inlet) and pushes fluid out of the nozzle 1045b, or vice versa if the pump is driven in reverse. This movement of fluid can enhance media flow in a culture vessel and / or can create turbulence to stimulate organoid development, etc. The use of the pump module 1009 can be suitable when gravity-driven flow is not suitable or is inefficient (e.g., due to properties of the media).

[0133] ​The housing 1032 can house any suitable electronic circuitry to enable wireless reception of power from the control circuit 1016 and operation of the pump 1044. The circuitry can include a power receiver 1035 to receive power wirelessly transmitted from the power antenna 1025, such as through inductive or capacitive coupling. The electronic circuitry can also include a power storage unit 1037 and a pump driver 1046.

[0134] FIG. 10 An example culture system 1100 is shown, which includes a pair of pump modules 1009a and 1009b housed in a culture vessel 1102 (see also FIG. 1 ). The vessel 1102 includes a culture chamber 1103 that houses an organoid 1121. Three compartments are located vertically above the culture chamber 1103, namely a pair of reservoirs 1105a and 1105b that hold respective culture media 1123a and 1123b, and a slot 1107 that houses a dummy module 1109, which is an example of the module 109 FIG. 12 . The reservoir 1105a communicates with the culture chamber 1103 via a pair of channels 1106a and 1106c, and the reservoir 1105b communicates with the culture chamber 1103 via a pair of channels 1106b and 1106d.

[0135] The pumps 1009a and 1009b are driven by power transmitted from the power antennas 1025a and 1025b of the control circuit 1016, respectively. The pump 1009a drives culture medium 1123a from the reservoir 1105a through the channel 1106c and into the culture chamber 1103. The pump 1009b drives culture medium from the culture chamber 1103 through the channel 1106d into the reservoir 1105b. Thus, there is a net pump-driven flow of medium from the reservoir 1105a to the reservoir 1105b. The pumps can be driven in reverse to move medium from the reservoir 1105b back to the reservoir 1105a, or this can occur via gravity-driven flow through the channels 1106a and 1106b.

[0136] FIG. 11 A culture system 1200 is shown that mechanically stimulates an organoid 1221 or other multicellular structure. The culture system 1200 includes a vessel 1102 (see FIG. 8 ), a control circuit 1216, and a pair of magnetic modules 1209a and 1209b housed in the culture chamber 1103 of the vessel 1102. The organoid 1221 can be located between the magnetic modules. The magnetic modules can function as magnetic actuators to apply an alternating mechanical strain to the organoid, with the driving principle being a magnetic force applied by the control circuit 1216 to produce linear driving.

[0137] The electrode module 809 can be located in the slot portion 1107 with two needle electrodes 834b extending into the organoid 1221 (see also FIG. 13 However, as described below, the mechanical stimulation of the organoid 1221 provided by movement of the magnetic modules 1209a and 1209b driven by the control circuit 1216 does not require the presence of the electrode module 809.

[0138] Each of the magnetic modules 1209a and 1209b includes a permanent magnet 1247, which can be encapsulated by a housing 1232. The housing can have a surface coating that facilitates attachment to the organoid 1221. The magnets 1247 of the magnetic modules 1209a and 1209b each have a north pole (N) and a south pole (S) with a magnetic axis extending through the two poles. The magnetic modules 1209a and 1209b can be arranged in the culture chamber 1103 so that the magnetic axes of the magnetic modules are coaxial with each other and anti-parallel (as shown) to produce a magnetic repulsion force, or parallel to produce a magnetic attraction force.

[0139] The control circuit 1216 includes at least one coil, such as a pair of fixed coils 1226a and 1226b, which can be energized to produce one or more additional magnetic fields. The one or more additional magnetic fields effectively enhance or diminish the attractive or repulsive force between the magnetic modules 1209a and 1209b, thereby driving the magnetic modules to move toward or away from each other. Each of the coils 1226a and 1226b defines a coil axis, which can be oriented parallel to the magnetic axes of the magnetic modules 1209a and 1209. FIG. 14 Energization of the two coils 1226a and 1226b is indicated by current arrows 1348, and the resulting magnetic field polarities at the coils are shown. As indicated by the motion arrows at 1349, the magnetic repulsion between the coil 1226a and the magnetic module 1209a and the magnetic repulsion between the coil 1226b and the magnetic module 1209b drive the magnetic modules to move toward each other, which will exert compression on the organoid 1221. In other cases, energization of the coils 1226a and 1226b can drive the magnetic modules 1209a and 1209b farther apart, which can exert tension on the organoid if attached to the two modules. In yet other cases, the magnetic modules can be used for magnetic sensing of organoid movement. The ability to exert repeated mechanical stress by the magnetic modules is particularly valuable for certain types of organoids, such as bone and muscle organoids, which can require such stress for normal development.

[0140] FIG. 15 and FIG. 12 How the culture system 1400 can be implemented in a culture system 1400 including a row of culture vessels 1402a-c is shown schematically FIG. 13 and FIG. 14magnetic drive mechanism. (Dotted arrows roughly indicate the location of the culture vessels, but the vessels themselves are omitted to simplify the illustration.) As described above for the single culture vessel 1102, each of the culture vessels 1402a-1402c houses a respective organoid 1421a-1421c between a pair of magnetic modules 1209a and 1209b. The control circuit 1416 provides a series of energizable coils 1426a-1426d arranged along the same line as the vessels 1402a-1402c, the organoids 1421a-1421c, and each pair of magnetic modules 1209a and 1209b. The coil axis defined by each of the coils 1426a-1426d extends along the vessels 1402a-1402c.

[0141] In the absence of energization of the coils 1426a-1426d FIG. 15 In the absence of energization of the coils 1426a-1426d FIG. 15 The positions of the magnetic modules 1209a and 1209b are shown in the absence of energization of the coils 1426a-1426d FIG. 12 Energization is indicated by arrows at 1448 in the absence of energization of the coils 1426a-1426d. As described above for the single culture vessel in FIG. 13 and FIG. 16 In the absence of energization of the coils 1426a-1426d

[0142] FIG. 14 is a schematic top view of a culture system 1600 having energizable coils 1626 of different configurations to drive FIG. 15 and FIG. 17The magnetic modules 1209a and 1209b move. (Only a subset of the coils 1626 is identified with a numerical identifier.) The culture system 1600 includes rows of containers 1602a-c. (The locations of the containers are roughly indicated with dashed arrows, but the containers themselves are omitted to simplify the illustration.) As described above for the culture system 1400, each of the containers 1602a-c houses a respective organoid 1621a-c between a pair of magnetic modules 1209a and 1209b. However, as shown, the magnetic axes of each pair of magnetic modules 1209a and 1209b are oriented orthogonal to the rows of containers 1602a-c, such as horizontal. The control circuit 1616 provides at least one row of coils 1626 or one pair of one row of coils 1626. Each coil 1626 can define a coil axis parallel to the magnetic axis of the magnetic modules. Proper energization of the coils 1626 linearly drives the magnetic modules 1209a and 1209b in a direction parallel to the rows of containers 1602a-c to mechanically stimulate the organoids 1621a-c.

[0143] Passive Module An example light module 1709 (i.e., an electrical / magnetic embodiment of the module 109) is shown configured to be wirelessly powered and controlled by the control circuit 1716 via a power antenna 1725 of the control circuit 1716. The light module 1709 includes a housing 1732 that houses a power receiver 1735 to receive power from the power antenna 1725, a power storage unit 1737, and a light source 1750. The light source generates light radiation that can be used for optical stimulation of a multicellular structure in a container (e.g., via optogenetics) and / or for illumination for optical detection of the multicellular structure.

[0144] Example light sources include light-emitting diodes, lasers, and the like. The light source can also include any suitable optics to direct or focus the light. For example, the light source can have a waveguide such as an optical fiber to direct light from the light module 1709 onto a surface of a multicellular structure and / or into the multicellular structure for internal illumination of the multicellular structure.

[0145] The light module 1709 can be used to facilitate imaging of a multicellular structure housed in a culture chamber of a culture container. For example, the light module can provide brightfield or darkfield illumination of a multicellular structure when the light module is suitably positioned in the culture container, such as in a storage portion, a trough portion, or a culture chamber of the culture container.

[0146] IV. FIGS. 18-20

[0147] This section describes example passive modules for use in the culture systems and methods of the present disclosure; see FIG. 18The passive modules are configured to operate without a power source and without interacting with control circuitry. Each passive module can be configured to be placed in a particular compartment of a container or alternatively can be placed in each of two or more compartments of a container.

[0148] FIG. 1A is a schematic view of an exemplary scaffold module 1809, which is an embodiment of the module 109 of FIG. 19 . 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 can be formed on the body 1851, such as by 3D printing, or the scaffold can be formed first and then attached to the body 1851. In either case, the scaffold 1822 can be mounted on any suitable side of the body 1851, such as a bottom side, a top side, or a lateral side. The scaffold module 1809 can be positioned in a trough of a culture container, such as during manufacture of the culture container or by a user. As further described in Section V below, in some cases the scaffold module can be an insert that forms one or more reservoirs of a culture container. The scaffold module can be used to mount and support a scaffold, as well as to hold the scaffold in place, and to enable improved handling and scaffold addition and removal.

[0149] FIG. 1A is shown an exemplary permeable interface module 1909, which is an embodiment of the module 109 of FIG. 19 . A cross-section of the permeable interface module is shown in FIG. 20 to show the internal structure of the permeable interface module. The permeable interface module 1909 has a hollow body 1951 defining a cavity 1952 that is in communication with an inlet 1953 and an interface opening 1954. The inlet 1953 can be plugged with a removable cap 1955. A permeable member 1956, such as a permeable membrane or gel, is attached to the hollow body 1951 at the interface opening 1954 to create a permeable interface 1957 (e.g., a permeable wall) on any suitable side of the body 1951, such as a bottom side, a top side, or a lateral side. The permeable interface 1957 allows fluid and / or small molecules to be selectively passed into or out of the cavity 1952 via the permeable interface.

[0150] The permeable interface module 1909 can house any suitable medium in the cavity 1952. The medium can be a gas, a liquid, a gel, etc. The medium can have a different phase, composition, and / or chemical potential than the culture medium (medium) present in the culture chamber of the vessel. The permeable interface 1957 can be positioned in physical contact with the multicellular structures and / or culture medium in the vessel. As an example, the permeable interface module 1909 can house air (or other gas) in the cavity 1952 to create a gas / liquid interface at the permeable member 1956, enabling culture of lung organoids in the culture chamber of the culture vessel. In other examples, the permeable interface module 1909 can house any suitable compound that can be discharged to the culture chamber of the culture vessel through the permeable member 1956.

[0151] FIG. 11 A permeable interface module 1909 is shown operatively positioned in the trough portion 1107 of the vessel 1102 (see also Container Assembly ), adjacent to the well 1108 and above the lung organoids 2021. The cavity 1952 is filled with gas such that the permeable member 1956 forms a gas-liquid interface between the gas in the cavity 1952 and the liquid culture medium in the culture chamber 1103.

[0152] A passive module housed in a compartment of a culture vessel can be described as a dummy module. The dummy module can be a placeholder. As with any module of the present disclosure, the dummy module can serve to reduce the hold-up volume of the vessel, and / or can obstruct or seal the trough portion above the culture chamber. However, the dummy module can also provide a surface shape (concave or convex), a surface chemistry / texturing (e.g., hydrophilicity, microstructuring, etc.), and / or a functional surface that facilitates a culture protocol.

[0153] V. FIGS. 21-40

[0154] This section describes exemplary vessel assemblies for use in the culture systems and methods of the present disclosure, vessel arrays formed from the vessel assemblies, and culture vessels of the vessel assemblies; see FIG. 21 .

[0155] FIG. 21 An exemplary vessel array 2101 is shown that illustrates a corresponding arrangement for culturing multicellular structures such as organoids. The vessel array 2101 includes at least one vessel strip 2118 held by a frame 2119. FIG. 22Only one container strip 2118 is shown, but the frame 2119 is configured to removably hold two, three, or more container strips 2118, which can be substantially identical to each other. In the illustrated embodiment, the frame 2119 defines eight receiving sites 2158, corresponding to the number of container strips 2118 arranged in a row, but the container strips 2118 can occupy only a subset of the receiving sites 2158 at any given time. The frame 2119 can have a footprint corresponding to that of a standard microplate (standard microplate footprint 127.71 mm x 85.43 mm), where the length and width of the footprint of the frame are within 10% or 5% of the length and width of a standard microplate. This correspondence of the frame 2119 to the standard microplate footprint enables mechanical compatibility with incubators, analytical instruments, and processing systems used for standard microplates.

[0156] The container strip 2118 forms a row of at least two, three, or more culture vessels, such as the four culture vessels 2102a-d in the illustrated embodiment. Each of the culture vessels 2102a-d includes a respective culture chamber 2103 formed by a lower region of the culture vessel.

[0157] FIG. 24 The container strip 2118 is shown removed from the frame 2119 to enable imaging of the multicellular structures contained in the culture chambers 2103. Removing the container strip 2118 from the frame 2119 allows the container strip to be properly positioned relative to the imaging system 2112 (or other detection system) without interference from the frame 2119 or other container strips 2118 held by the frame.

[0158] 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 transmissive to optical radiation, such as visible light, and can have smooth inner and outer surfaces that can optionally be planar to minimize scattering of the optical radiation. In the illustrated embodiment, each culture chamber 2013 has a bottom optical window 2159a, and a pair of lateral optical windows 2159b and 2159c arranged opposite each other through the culture chamber 2103 (see also FIG. 26 , FIG. 30 and FIG. 22 ).

[0159] FIG. 21Imaging system 2112 is shown as capturing images of a multicellular structure housed in culture vessel 2102c. A thin section of the multicellular structure can be illuminated through a lateral optical window 2159b (and / or window 2159c) of the culture vessel using a light source 2114 that produces a light sheet 2160. Light (e.g., fluorescence) from the multicellular structure can be collected with an objective 2161 that collects light through a bottom optical window 2159a of culture vessel 2102c. Thus, imaging system 2112 can perform a selective plane illumination microscope (SPIM), also known as light sheet microscopy. In other examples, illumination can be performed through bottom optical window 2159a and light collected from one of lateral optical windows 2159b and 2159c. In still other examples, illumination can be performed with a light module located in the culture vessel (e.g., see Section III). In additional other examples, imaging system 2112 can utilize a two-photon excitation microscope, tomography, etc.

[0160] Container strip 2118 provides a respective pair of reservoirs 2105a and 2105b and a respective trough 2107 above culture chamber 2013 for each of vessels 2102a-d (see FIG. 22 and FIG. 23 ). A trough module 2109 can be located in trough 2107 and can be any of the electrical / magnetic modules and passive modules disclosed herein (e.g., see Sections I, III, and IV).

[0161] FIG. 23 An exploded view of container strip 2118 is shown. The container strip includes a housing 2162 (interchangeably referred to as a shell), a plurality of inserts 2163 configured to be housed in housing 2162, and a cover 2164 configured to cover an open top side of housing 2162 and / or inserts 2163. In some embodiments, cover 2164 can not be needed. A respective pair of magnetic modules 1209a and 1209b can be located in housing 2162 beneath each insert 2163 (see also Section III), although any other suitable module or modules can be located in the housing beneath the inserts, or no modules can be located in the housing beneath the inserts.

[0162] The interior of housing 2162 can be divided into a plurality of sections, such as sections 2165a-d. Sections 2165a-d can be integrally formed with one another. Sections 2165a-d can be directly attached to one another, or can be separated from one another with a respective spacing region 2166 located intermediate each pair of adjacent sections.

[0163] Each section 2165a-d of the housing 2162 includes a lower region forming a receptacle 2167 and an upper region forming a receiving space 2168 of one of the insert portions 2163 (see FIG. 26 , FIG. 28-30 and FIG. 26 ). The housing 2162 and one of the insert portions 2163 are used to cooperatively form each culture chamber 2103, which can capture one or more modules in the culture chamber, such as the pair of magnetic modules 1209a and 1209b in the illustrated embodiment (see FIG. 26 ). The receptacle 2167 provides a lateral wall of the culture chamber 2103, and one of the insert portions 2163 provides a top wall portion of the culture chamber. A scaffold for an organoid can be provided in the receptacle 2167 of each insert portion 2163, such as formed in or placed in (e.g., attached to a bottom or lateral wall of the receptacle) prior to assembly of the insert portion 2163 with the housing 2162. In other cases, the scaffold can be provided on a bottom side of the insert portion 2163, such as 3D printed on the insert portion 2163 or attached to the insert portion 2163 after the scaffold is formed, prior to assembly of the insert portion with the housing 2162. In yet other cases, the scaffold can be provided by the module 2109 (see also Section IV).

[0164] Each insert portion 2163 is configured to fit into one of the sections 2165a-d. Accordingly, the outer dimensions of the insert portion can correspond to the dimensions of the receiving space 2168. The receptacle 2167 can have one or more horizontal dimensions that are less than the receiving space 2168 to form a shoulder 2169 at the bottom of the receiving space 2168 to support the insert portion 2163 and prevent further advancement down into the section (see FIG. 29 , FIG. 30 and FIG. 27 ).

[0165] Each insert portion 2163 can be locked in the housing 2162 by a snap-fit mechanism that engages when the insert portion is in the housing (see FIG. 31-33 and FIG. 33 ). The insert portion can define a protrusion 2170 that is received in a corresponding opening 2171 defined by a lateral wall of the housing 2162, or the insert portion can define an opening and the housing can define a protrusion.

[0166] Each insert portion 2163 defines various openings at its bottom. The openings include at least one channel at the bottom of each storage portion 2105a and 2105b, such as the pair of channels 2106a and 2106b (see FIG. 26). Each of the channels 2106a and 2106b extending from the storage portion 2105a or 2105b provides fluid communication between one of the storage portions and the culture chamber 2103 located vertically below the storage portion (see also FIG. 26 ). The insert portion also defines an aperture 2108 at the bottom end of the trough portion 2107 to provide fluid communication between the trough portion 2107 and the culture chamber 2103 (see also FIG. 33 、 FIG. 34 、 FIG. 35 ).

[0167] FIG. 21 A lid assembly 2172 of the container array 2101 is shown (see also FIG. 26 ). The lid assembly 2172 includes a series of lids 2164 to cover a corresponding number of container strips 2118 held by the frame 2119 (see also FIG. 36 ). Each lid 2164 can fit over the top of a container strip (e.g., the top of the housing 2162) and can have a peripheral flange that projects downward and is configured to overlap the container strip in the vertical direction at the top edge of the container strip and to horizontally surround the container strip. Maintaining a sterile condition can be a major concern throughout the culturing and testing, which can take several months. Thus, it would be advantageous for the lid assembly 2172 to block contaminating microorganisms from entering through the open top and trough portion 2107 of the storage portion 2105a and 2015b of each of the containers 2102a-d of each container strip 2118. The lid assembly 2172 allows all of the container strips to be covered by the same lid assembly, but individual lids 2164 can be removed from the lid assembly 2172. For example, the lid assembly 2172 can include a carrier 2173 to which each lid 2164 is removably attached (e.g., via a weak adhesive, an interference fit, or a snap fit mechanism, etc.). Such a construction enables any individual container strip 2118 including a lid 2164 to be easily removed from other container strips 2118 of the container array 2101 and the lids 2164 of the other container strips 2118 for, e.g., imaging or other processing. In other examples, the carrier 2173 can be omitted and the lids 2164 can be integrally formed with one another and configured to be detachable by breaking a frangible connection that joins adjacent pairs of lids 2164 to one another.

[0168] FIG. 37 and FIG. 23 Another example insert portion 3663 placed in the housing 2162 is shown (see also FIG. 38Except that the insertion portion 3663 includes a gasket 3674 configured to create a fluid seal with the housing 2162, the insertion portion 3663 is identical to the insertion portion 2163. The gasket 3674 may be formed of a softer, more deformable material (e.g., an elastomer) and may be attached to the body 3675, which is formed of a harder, less deformable material. In some cases, the gasket 3674 may be formed on the body 3675 by overmolding. The gasket 3674 may be located 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, etc.

[0169] FIG. 23 Another exemplary insertion portion 3863 is shown placed in the housing 2162 (see also...) FIG. 33 The insertion portion 3863 forms a pair of storage portions 3805a and 3805b, which are adjacent to each other and share a transverse wall, rather than being separated by a central slot (with). FIG. 39 (Compared to). If the scaffold is to be attached to the bottom side of the insert, the insert 3863 may be advantageous because there is more surface area available for attachment. Furthermore, the insert 3863 allows the channel at the bottom of the insert to be positioned more centrally for vertical alignment with multicellular structures centrally located in the culture chamber below the insert.

[0170] FIG. 23 Another exemplary insertion portion 3963 is shown placed in housing 2162 (see also...) FIG. 33 The insertion section 3963 forms four storage sections 3905a to 3905d and a central slot 3907 located between the storage sections (and...). FIG. 40 (Compared to). Insertion 3963 may be advantageous when feeding the interior and exterior of multicellular structures with different culture media.

[0171] FIG. 23 Another exemplary insertion portion 4063 is shown placed in the housing 2162 (see also...) Selected Aspects Similar to insertion part 3963, insertion part 4063 forms four storage parts 4005a to 4005d, but lacks the central slot located between the storage parts (and). ​ (Compared to). Insertion section 4063 combines the potential advantages of having more than two storage sections and centrally positioning the channel above the multicellular structure.

[0172] VI. ​

[0173] This section will describe selected aspects of the systems, methods, and apparatus of this disclosure in the form of a series of index paragraphs.

[0174] Paragraph Al. A system for culturing a multicellular structure, such as an organoid, the system comprising: (a) a container comprising a culture chamber to house a multicellular structure, such as an organoid; (b) an electric / magnetic module configured to be coupled to and / or located in the container, optionally at a location in or adjacent to the culture chamber; and (c) a control circuit configured to wirelessly power and / or wirelessly operate the electric / magnetic module; wherein, optionally, the electric / magnetic module is removably coupled to and / or removably located in the container, wherein, optionally, the electric / magnetic module is capable of being housed in each of two or more compartments of the container, and wherein, optionally, the two or more compartments are selected from the culture chamber, one or more storage portions, and / or slot portions.

[0175] Paragraph A2. The system of paragraph Al, wherein the control circuit is configured to wirelessly power and / or wirelessly operate the electric / magnetic module using near-field radiation.

[0176] Paragraph A3. The system of paragraph A2, wherein the control circuit is configured to wirelessly transmit electrical power to the electric / magnetic module via inductive coupling or capacitive coupling.

[0177] Paragraph A4. The system of paragraph A2 or A3, wherein the control circuit is configured to wirelessly communicate with the electric / magnetic module via at least one near-field communication protocol.

[0178] Paragraph A5. The system of any one of paragraphs Al to A4, wherein the electric / magnetic module is housed in or configured to be housed in the culture chamber and comprises a magnet, and wherein the control circuit is configured to generate a magnetic field that drives the magnet to move in the culture chamber.

[0179] Paragraph A6. The system of paragraph A5, the electric / magnetic module being a first module and the magnet being a first magnet, further comprising a second module comprising a second magnet, wherein the first module and the second module are simultaneously housed in or configured to be simultaneously housed in the culture chamber, and wherein the control circuit is configured to drive the first magnet and the second magnet to move relative to each other, optionally towards each other and / or away from each other, within the culture chamber.

[0180] Paragraph A7. The system of any one of paragraphs Al to A6, wherein the electric / magnetic module comprises a sensor.

[0181] Paragraph A8. The system of paragraph A7, wherein the electric / magnetic module comprises a chemical sensor, an electrical sensor, an optical sensor, and / or a temperature sensor.

[0182] Paragraph A9. The system of any one of paragraphs Al to A8, wherein the electric / magnetic module comprises an electrode.

[0183] Paragraph A10. The system of paragraph A9, wherein the electrode is configured to electrically stimulate and / or electrically sense the multicellular structure in the culture chamber.

[0184] Paragraph A11. The system of any one of paragraphs A1-A10, wherein the electro / magnetic module comprises a light source.

[0185] Paragraph A12. The system of paragraph A11, wherein the light source is configured to illuminate at least a portion of the multicellular structure in the culture chamber.

[0186] Paragraph A13. The system of any one of paragraphs A1-A12, wherein the electro / magnetic module comprises a pump configured to drive fluid into and / or out of the culture chamber.

[0187] Paragraph A14. The system of any one of paragraphs A1-A13, wherein the electro / magnetic module is located in or configured to be located in a slot portion adjacent to the culture chamber, and wherein, optionally, the container defines a hole providing fluid communication between the slot portion and the culture chamber.

[0188] Paragraph A15. The system of paragraph A14, wherein the container comprises two or more storage portions in fluid communication with the culture chamber, wherein, optionally, the slot portion is located vertically above the culture chamber, and, optionally, between at least one pair of the two or more storage portions, wherein, optionally, each of the two or more storage portions is in communication with the culture chamber, respectively, wherein, optionally, each of the two or more storage portions is not connected to any external source of liquid, such as via a tube, wherein, optionally, each of the two or more storage portions and the culture chamber are formed from and / or located in the same housing, and wherein, optionally, each of the two or more storage portions is directly attached to the culture chamber and / or shares a wall with the culture chamber.

[0189] Paragraph A16. The system of paragraph A14 or A15, wherein the electro / magnetic module comprises in a group of two or more modules configured to perform different functions from each other and coupled with and / or located in the container, and, optionally, interchangeably located in the same compartment of the container, such as in the same storage portion, culture chamber, and / or slot portion.

[0190] Paragraph A17. The system according to any one of paragraphs Al to A16, wherein the vessel comprises at least one or two or more storage sections, each storage section being located above (optionally vertically above) the culture chamber, and optionally each storage section shares a wall with the culture chamber, wherein optionally the two or more storage sections are integrally formed with each other and / or by the same insert, and wherein optionally the electric / magnetic module is located in the vertical direction intermediate the top side of the two or more storage sections and the bottom of the culture chamber, and / or the system further optionally comprises a removable lid configured to be placed on the vessel to cover the at least one or two or more storage sections.

[0191] Paragraph A18. The system according to any one of paragraphs Al to A17, further comprising a vessel assembly forming a vessel row connected to each other and comprising vessels, each vessel in the vessel row comprising a respective culture chamber to contain multicellular structures (such as organoids), wherein optionally the vessels in the vessel row are substantially identical to each other, and wherein optionally the electric / magnetic module is movable among the vessels in the vessel row.

[0192] Paragraph A19. The system according to paragraph A18, wherein the vessel assembly has a length corresponding to the length or width of a standard microplate footprint.

[0193] Paragraph A20. The system according to paragraph A18 or A19, further comprising a vessel array comprising the vessel assembly and forming at least two or three rows of vessels, wherein each vessel in the at least two or three rows of vessels comprises a respective culture chamber to contain multicellular structures.

[0194] Paragraph A21. The system according to any one of paragraphs A18 to A20, further comprising a frame to hold a plurality of vessel assemblies comprising the vessel assembly, the frame optionally having a length and / or width corresponding to the length and / or width of a standard microplate footprint, wherein optionally the plurality of vessel assemblies are substantially identical to each other, and wherein optionally the plurality of vessel assemblies comprise a respective plurality of housings that are substantially identical to each other.

[0195] Paragraph A22. The system according to any one of paragraphs Al to A21, wherein the vessel comprises an optical window formed by the culture chamber wall.

[0196] Paragraph A23. The system according to any one of paragraphs Al to A22, further comprising a scaffold located in or configured to be located in the culture chamber and configured to support organoid formation in the culture chamber.

[0197] Paragraph A24. The system according to any one of paragraphs Al to A23, wherein the vessel is provided by the apparatus of any one of paragraphs CI to C20.

[0198] Paragraph B1. A method of culturing a multicellular structure such as an organoid, the method comprising: (a) housing the multicellular structure in a culture chamber of a container, wherein an electro / magnetic module is removably coupled with and / or located in the container, optionally at a location in or adjacent to the culture chamber; and (b) wirelessly powering / wirelessly operating the electro / magnetic module using a control circuit, wherein the electro / magnetic module is optionally removably coupled with and / or removably located in the container, and wherein the electro / magnetic module is optionally capable of being housed in each of two or more compartments of the container, and wherein the two or more compartments are optionally selected from the culture chamber, one or more storage and / or slot portions, and wherein the multicellular structure is optionally an organoid having a diameter of at least 0.2, 0.5, 1, or 2 mm and is formed / grown in the culture chamber without connecting the container to an external source of liquid (e.g., culture medium) (e.g., via tubing and / or a tube) and / or without electrically connecting the container to wiring or other electrical conductors.

[0199] Paragraph B2. The method of paragraph B1, wherein the powering / operating comprises wirelessly transmitting electrical energy to the electro / magnetic module.

[0200] Paragraph B3. The method of paragraph B1 or B2, wherein the powering / operating is performed at least in part via inductive or capacitive coupling of the control circuit and the electro / magnetic module with each other.

[0201] Paragraph B4. The method of any of paragraphs B1-B3, wherein the powering / operating comprises wirelessly communicating with the electro / magnetic module using near-field radiation.

[0202] Paragraph B5. The method of paragraph B4, wherein the wirelessly communicating comprises exchanging data with the electro / magnetic module using at least one near-field communication protocol.

[0203] Paragraph B6. The method of any of paragraphs B1-B5, wherein the electro / magnetic module comprises a magnet, and wherein the powering / operating comprises using a magnetic field generated by the control circuit to drive the magnet to move in the culture chamber.

[0204] Paragraph B7. The method of paragraph B6, wherein the culture chamber houses a pair of electro / magnetic modules, each of which comprises a magnet, and wherein the powering / operating comprises driving the electro / magnetic modules relative to each other, and optionally, toward each other and / or away from each other, in the culture chamber.

[0205] Paragraph B8. The method of any of paragraphs B1-B7, wherein the powering / operating comprises using a sensor of the electro / magnetic module to sense a property of the multicellular structure and / or of a culture medium (e.g., located in the culture chamber) in contact with the multicellular structure.

[0206] Paragraph B9. The method of any one of paragraphs B1 to B8, wherein the powering / operating comprises electrically stimulating the multi-cellular structure.

[0207] Paragraph B10. The method of any one of paragraphs B1 to B9, wherein the powering / operating comprises driving fluid into and / or out of the culture chamber.

[0208] Paragraph B11. The method of any one of paragraphs B1 to B10, wherein the electric / magnetic module is located in the culture chamber.

[0209] Paragraph B12. The method of any one of paragraphs B1 to B11, wherein the electric / magnetic module is located in the container and at least predominantly (i.e. more than half by volume) outside the culture chamber.

[0210] Paragraph B13. The method of paragraph B12, wherein the electric / magnetic module is located in a trough portion defined by the container.

[0211] Paragraph B14. The method of any one of paragraphs B1 to B13, further comprising collecting data relating to the multi-cellular structure while the multi-cellular structure is held in the culture chamber.

[0212] Paragraph B15. The method of paragraph B14, wherein the collecting data comprises capturing an image of at least a portion of the multi-cellular structure.

[0213] Paragraph B16. The method of paragraph B14 or B15, wherein the collecting data is performed using a sensor of the module.

[0214] Paragraph B17. The method of any one of paragraphs B1 to B16, further comprising disposing the electric / magnetic module in a compartment of the container.

[0215] Paragraph B18. The method of paragraph B17, wherein the disposing comprises capturing the electric / magnetic module in the culture chamber.

[0216] Paragraph B19. The method of paragraph B17, wherein the disposing comprises placing the electric / magnetic module in the container but outside the culture chamber, optionally in a storage portion or a trough portion of the container.

[0217] Paragraph B20. The method of any one of paragraphs B17 to B19, further comprising, prior to the disposing, selecting the electric / magnetic module from a group of two or more differently functional modules located outside the container, the selection optionally being based on (i) a type of organoid present in the culture chamber and / or to be cultured, (ii) a culture protocol or protocol stage selected for the organoid, (iii) a test condition for the organoid, and / or (iv) a parameter sensed for the organoid and / or a culture medium in contact with the organoid.

[0218] Paragraph B21. The method of paragraph B20, further comprising selecting two or more differently functional modules from the group, wherein, for each selected module, the setting comprises coupling the selected module with the vessel and / or positioning the selected module in the vessel.

[0219] Paragraph B22. The method of paragraph B20 or B21, wherein the group of two or more differently functional modules comprises at least one passive module, and wherein the selecting comprises, optionally, selecting the passive module.

[0220] Paragraph B23. The method of any one of paragraphs B1 to B22, wherein the method is performed with the system of any one of paragraphs Al to A24.

[0221] Paragraph CI. An apparatus for culturing a multicellular structure, such as an organoid, the apparatus comprising: (a) a housing, optionally having an open top; and (b) an insert comprising at least one reservoir or two or more reservoirs, the insert configured to be housed in the housing, optionally via the open top, such that the housing and the insert cooperate to form a culture chamber for the multicellular structure, the culture chamber being located below (optionally vertically below) the two or more reservoirs and being in fluid communication with each of the at least one reservoir or the two or more reservoirs, optionally via a respective channel defined by the insert.

[0222] Paragraph C2. The apparatus of paragraph CI, further comprising a lid configured to be placed on the housing to cover the at least one reservoir and / or each of the two or more reservoirs.

[0223] Paragraph C3. The apparatus of paragraph C2, wherein the lid is configured to completely cover the open top of the housing.

[0224] Paragraph C4. The apparatus of any one of paragraphs CI or C3, further comprising a scaffold configured to support organoid formation in the culture chamber.

[0225] Paragraph C5. The apparatus of paragraph C4, wherein the scaffold is attached to a wall of the housing.

[0226] Paragraph C6. The apparatus of paragraph C4, the housing and the insert each being part of a vessel, further comprising a module providing the scaffold, wherein the module is configured to be coupled with (optionally removably) and / or located in a compartment of the vessel, such as a slot defined by the insert.

[0227] Paragraph C7. The apparatus of paragraph C6, wherein the slot is located vertically above the culture chamber and between at least one pair of the two or more reservoirs.

[0228] Paragraph C8. The apparatus according to any one of paragraphs C1-C7, wherein the housing comprises a section having an upper region defining a receiving space and a lower region forming a receptacle, wherein the insert portion is configured to be received in the receiving space, and wherein the receptacle is configured to cooperate with the insert portion to form the culture chamber.

[0229] Paragraph C9. The apparatus according to paragraph C8, wherein the insert portion is configured to form a fluid seal with the plurality of side walls and / or the bottom wall of the upper region of the section.

[0230] Paragraph C10. The apparatus according to paragraph C9, wherein the insert portion comprises a body and a gasket attached to the body, and wherein the gasket is configured to engage with each of the plurality of side walls and / or the bottom wall to form the fluid seal.

[0231] Paragraph C11. The apparatus according to paragraph C10, wherein the gasket is molded on the body.

[0232] Paragraph C12. The apparatus according to any one of paragraphs C1-C11, wherein the insert portion is formed with a slot portion and defines a hole at a bottom end of the slot portion, further comprising a module located in or configured to be located in the slot portion, the module being located vertically above the hole, and wherein, optionally, the module is an electrical / magnetic module.

[0233] Paragraph C13. The apparatus according to any one of paragraphs C1-C12, wherein the apparatus comprises a plurality of insert portions, and wherein the housing forms a row of sections, each section configured to receive a respective insert portion of the plurality of insert portions to cooperate with each section and respective insert portion to form the culture chamber for the multicellular structure.

[0234] Paragraph C14. The apparatus according to paragraph C13, wherein the sections of the row of sections are integrally formed as a single piece with each other.

[0235] Paragraph C15. The apparatus according to paragraph C13 or C14, further comprising a lid configured to be placed on the housing to cover the row of sections.

[0236] Paragraph C16. The apparatus according to any one of paragraphs C1-C15, wherein the lower region of the housing forms at least one optical window of the culture chamber.

[0237] Paragraph C17. The apparatus according to paragraph C16, wherein the lower region of the housing forms at least two optical windows of the culture chamber.

[0238] Paragraph C18. The apparatus according to paragraph C17, wherein the at least two optical windows comprise a bottom window and at least one lateral window.

[0239] Paragraph C19. The apparatus according to either of Paragraphs C17 or C18, wherein the at least two optical windows comprise a pair of lateral windows arranged opposite one another.

[0240] Paragraph C20. The apparatus according to any of Paragraphs C1-C19, wherein the housing and the insert each form part of a same container, further comprising one or more modules, each of which is configured to couple with and / or be located in the container, optionally a set of two or more different functional modules, such as a set comprising at least one or at least two electric / magnetic modules and / or at least one or at least two passive modules, optionally at least two of the different functional modules are configured to be interchangeably placed in a same compartment (e.g., a same storage, culture chamber, or trough) of the container.

[0241] Paragraph D1. A method of culturing a multicellular structure, such as an organoid, the method comprising: (a) placing an insert, optionally comprising at least one storage or two or more storages, in a housing to cooperate with the insert and the housing to form a culture chamber, the culture chamber being optionally located vertically below and in fluid communication with each of the at least one storage or two or more storages; and (b) culturing the multicellular structure in the culture chamber.

[0242] Paragraph D2. The method according to Paragraph D1, further comprising placing a lid on the housing to cover the two or more storages.

[0243] Paragraph D3. The method according to either of Paragraphs D1 or D2, wherein the culture chamber is in fluid communication with each of the two or more storages via a channel defined by the insert.

[0244] Paragraph D4. The method according to any of Paragraphs D1-D3, further comprising disposing a scaffold in the housing, wherein the scaffold is configured to facilitate organoid formation in the culture chamber, optionally embedding cells of the multicellular structure in the scaffold.

[0245] Paragraph D5. The method according to Paragraph D4, wherein the disposing comprises forming the scaffold in the housing, optionally prior to placing the insert.

[0246] Paragraph D6. The method according to Paragraph D4, wherein the disposing comprises placing a pre-formed scaffold in the housing.

[0247] Paragraph D7. The method according to Paragraph D6, wherein the pre-formed scaffold is placed in the housing prior to placing the insert.

[0248] Paragraph D8. The method of paragraph D6, wherein placing the pre-shaped scaffold in the housing is performed by placing the insert or after placing the insert, and wherein, optionally, the pre-shaped scaffold is already attached to the bottom side of the insert before placing the insert.

[0249] Paragraph D9. The method of any one of paragraphs D1-D8, wherein the housing comprises a segment row, wherein placing comprises placing at least two inserts in the segment row to form two or more separate culture chambers, and wherein culturing the multicellular structure comprises culturing a respective organoid in each of the two or more separate culture chambers.

[0250] Paragraph D10. The method of any one of paragraphs D1-D9, further comprising collecting data related to the multicellular structure while the multicellular structure remains in the culture chamber.

[0251] Paragraph D1. The method of any one of paragraphs D1-D10, wherein collecting data comprises capturing an image of at least a portion of the multicellular structure.

[0252] Paragraph D12. The method of any one of paragraphs D1-D11, wherein the method is performed using any of the devices of paragraphs C1-C20.

[0253] Paragraph E1. A system for culturing a multicellular structure, such as an organoid, comprising: (a) a vessel comprising a culture chamber to house the multicellular structure, and at least one reservoir or at least two or more reservoirs and optionally a slot portion each in fluid communication with the culture chamber; and (b) two or more modules having different functions from one another and configured to interchangeably couple with and / or be disposed in the vessel.

[0254] Paragraph E2. The system of paragraph E1, wherein the vessel defines a hole in communication with the culture chamber at a bottom end of the slot portion.

[0255] Paragraph E3. The system of paragraph E2, wherein each of the two or more modules is configured to be positioned adjacent to the hole when placed in the slot portion.

[0256] Paragraph E4. The system of any one of paragraphs E1-E3, wherein the two or more modules comprise a first module having a permeable membrane.

[0257] Paragraph E5. The system of paragraph E4, wherein the membrane is configured to form an interface between a liquid in the culture chamber and a liquid or a gas in the first module.

[0258] Paragraph E6. The system of any one of paragraphs E1-E5, wherein at least one of the two or more modules is an electrical / magnetic module.

[0259] Paragraph E7. The system of paragraph E6, wherein the at least one module comprises a module having an electronic device.

[0260] 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.

[0261] Paragraph E9. The system of any of paragraphs El to E8, wherein the container is provided by the apparatus of any of paragraphs CI to C20.

[0262] Paragraph Fl. A method of culturing a multicellular structure, such as an organoid, the method comprising: (a) housing the multicellular structure in a culture chamber of a container, wherein the container defines a trough portion in communication with the culture chamber, and wherein a first module is positioned in the trough portion; (b) removing the first module from the trough portion; and (c) placing a second module in the trough portion, wherein the second module is configured to perform a different function than the first module.

[0263] Paragraph F2. The method of paragraph Fl, wherein the container defines an aperture providing communication between the trough portion and the culture chamber.

[0264] Paragraph F3. The method of paragraph Fl or F2, wherein at least one of the first module and the second module extends from the trough portion into the culture chamber via the aperture.

[0265] Paragraph F4. The method of any of paragraphs Fl to F3, wherein at least one of the first module and the second module comprises an electronic device.

[0266] Paragraph F5. The method of any of paragraphs Fl to F4, wherein a module of the first module and the second module comprises a membrane forming an interface between a liquid in the culture chamber and a liquid or a gas in the module.

[0267] Paragraph F6. The method of any of paragraphs Fl to F5, wherein at least one of the first module and the second module comprises an electrode in contact with the multicellular structure and / or the liquid in the culture chamber.

[0268] Paragraph F7. The method of any of paragraphs Fl to F6, further comprising wirelessly powering / wirelessly operating one of the first module and the second module using a control circuit while the one of the first module and the second module is positioned in the trough portion.

[0269] Paragraph F8. The method of any of paragraphs Fl to F7, wherein the container is provided by the apparatus of any of paragraphs CI to C20.

[0270] Paragraph G1. An organoid culture vessel that does not require connection of tubes and wiring to the vessel itself.

[0271] Paragraph H1. A method of organoid culture comprising forming / growing an organoid (e.g., a large organoid) in a vessel without connecting the vessel to an external source of liquid (e.g., via one or more tubes such as tubing) and / or without electrically connecting the vessel to an external electrical conductor (e.g., an electrical conductor that is electrically connected to an electrical / magnetic device and / or control circuitry).

[0272] While one or more applications have been described above with the aid of example and further information provided by the drawings and the claims, persons having ordinary skill in the art will appreciate that many other variations, combinations, and modifications of the examples and features described herein can be made without departing from the inventive concepts disclosed herein. Furthermore, one or more applications should not be considered to be limited to any particular purpose or example described herein, but should be considered to be applicable to achieving a variety of purposes. The disclosure describes some examples of the present technology with reference to the accompanying drawings, in which only some possible examples are shown. Other aspects can be implemented, however, in many different forms and should not be deemed to be limited to the examples described herein, even if not explicitly shown in combination. Rather, these examples are provided so that the present disclosure will be thorough and complete, and will fully convey the scope of the possible examples to those skilled in the art.

Claims

1. A system for culturing multicellular structures, the system comprising: A container, comprising a culture chamber configured to contain the multicellular structure; An electro / magnetic module configured to be located in the container, in or adjacent to the culture chamber, the electro / magnetic module including a magnet, electrodes, sensors, actuators, pumps or light sources; as well as A control circuit configured to wirelessly power and / or wirelessly operate the electro / magnetic module; A container assembly forming rows of containers, the rows of containers being interconnected and including the containers, each container in the rows of containers including a corresponding culture chamber to accommodate multicellular structures; and A frame that holds a plurality of container assemblies including the container assembly, the frame having a length and / or width corresponding to the length and / or width of the occupancy area of ​​a standard microplate.

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

3. The system according to claim 1, wherein, The control circuit is configured to wirelessly transmit electrical power to the electro / magnetic module via inductive or capacitive coupling.

4. The system according to claim 1, wherein, The electro / magnetic module is housed in or configured to be housed in the culture chamber and includes a magnet, wherein the control circuitry is configured to generate a magnetic field that drives the magnet to move within the culture chamber.

5. The system according to claim 1, wherein, The electro / magnetic module includes a chemical sensor, an electrical sensor, an optical sensor, and / or a temperature sensor.

6. The system according to any one of claims 1 to 5, wherein, The electro / magnetic module includes a pump configured to drive fluid into and / or out of the culture chamber.

7. The system according to any one of claims 1 to 5, wherein, The electro / magnetic module is located or configured to be located in a tank adjacent to the culture chamber.

8. The system according to claim 7, wherein, The container is defined with an orifice providing fluid communication between the tank and the culture chamber.

9. The system according to claim 7, wherein, The container includes two or more storage sections in fluid communication with the culture chamber, wherein the tank section is located vertically above the culture chamber.

10. The system according to claim 9, wherein, The slot is located between at least one pair of storage sections among the two or more storage sections.

11. The system according to claim 9, wherein, Each of the two or more storage sections is connected to the culture chamber.

12. A method for culturing multicellular structures, the method comprising: A multicellular structure is contained in a culture chamber of a container, wherein an electro / magnetic module is located in the container and in or adjacent to the culture chamber, the electro / magnetic module including a magnet, an electrode, a sensor, an actuator, a pump or a light source, wherein the container is contained in a container assembly forming a row of containers connected to each other; Wireless power supply / operation of the electro / magnetic module using control circuitry; and A frame holds multiple container assemblies including the container assembly, the frame having a length and / or width corresponding to the length and / or width of the occupancy area of ​​a standard microplate.

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

14. The method according to claim 12, wherein, Power supply / operation includes wirelessly transmitting electrical energy to the electro / magnetic module.

15. The method according to claim 12, wherein, Power supply / operation is performed at least in part via inductive or capacitive coupling between the control circuit and the electro / magnetic module.

16. The method according to claim 12, wherein, The electro / magnetic module includes a magnet, and the power supply / operation includes using a magnetic field generated by the control circuit to drive the magnet to move within the culture chamber.

17. The method according to any one of claims 12 to 16, wherein, Power supply / operation includes using sensors from the electro / magnetic module to sense the properties of the multicellular structure and / or culture medium in the culture chamber.

18. The method according to any one of claims 12 to 16, wherein, Power supply / operation includes driving fluid inflow and / or outflow from the culture chamber.

19. The method according to any one of claims 12 to 16, further comprising collecting data related to the multicellular structure while the multicellular structure is held in the culture chamber.

20. The method according to claim 19, wherein, The module's sensors are used to collect data.

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