Microfluidic device and method for forming multicellular constructs of selected shapes

Abstract

A microfluidic device for forming multicellular constructs of selected shapes. The microfluidic device comprises a bottom layer including a plurality of isolated open molds of selected shapes each having a top opening and a hydrophobic surface. The microfluidic device also comprises a top layer including a fluid channel extending from an inlet to an outlet, the fluid channel including an open channel having a bottom opening, the top layer slidably coupled to the bottom layer and movable among a plurality of positions relative to the bottom layer. The one of the plurality of positions, the top opening of the microfluidics device may be uncovered by the top layer to allow selective removal of a multicellular construct formed in the open mold having a selected shape from the open mold through the top opening.

Description

MICROFLUIDIC DEVICE AND METHOD FOR FORMING MULTICELLULAR CONSTRUCTSOF SELECTED SHAPESRELATED APPLICATIONS

[0001] This claims priority from United States provisional patent application no. 63 / 689,164, filed August 30, 2024, the entire contents of which are incorporated herein by reference.FIELD

[0002] This disclosure relates to microfluidics and related methods for producing cell aggregates, particularly to microfluidics devices and methods for forming multicellular constructs or other organoids of selected shapes.BACKGROUND

[0003] Organoids are three-dimensional (3D) constructs of self-organized cell aggregates that recapitulate many aspects of the complex structures, functions, and cellular complexity of human organs and tissues, and mimic their in vivo physiology. Organoids include tumoroids and can have various external shapes. Organoids may reproduce patient-specific intratumor heterogeneity, gene and protein expression, molecular signatures, metabolic activity, and tissue-like microstructures. Tumoroids refer to organoids that are derived from or replicate tumors or cancer tissues, and may be prepared using cells from primary tumors harvested from patients. Tumoroids can recapitulate complex genetic and molecular compositions of solid cancers. They can be maintained and analyzed in vitro but are useful for predicting in vivo biology.

[0004] Multicellular, patient-derived organoids, such as multicellular tumoroids, find many applications in medical research, drug discovery, and regenerative medicine. These applications may require complex organoid models, including organoids with diverse morphologies. Microfluidics-based cell culture platforms enable time-efficient confined organoid generation.

[0005] Microfluidics refers to miniaturized fluid-handling systems or devices for processing and manipulating extremely small volumes (on the order of 10'12to 10'3microliters) of fluids using fluid channels that include microchannels (on the order of 10° to 103micrometers). Fluids behave differently at the microscale due to the smaller dimensional sizes, as compared to at the macroscale, because various factors such as surface tension, energy dissipation, fluidic resistance, and diffusion may become dominate in a microfluidic system.

[0006] Microfluidics devices have been used for generation and handling of multicellular spheroids.SUMMARY

[0007] It is desirable to form multicellular constructs, such as organoids or tumoroids, in an efficient and user-friendly manner, such that multiple multicellular constructs may be formed at once in a single device. However, it is also desirable to allow for the addressable extraction of one or more multicellular constructs from the device at a time, and for these multicellular constructs to be extracted intact, e.g., to extract an individual aggregate of cells with its extracellular matrix attached.

[0008] It is also desirable to form multicellular constructs with different shapes and to achieve a subsequent transfer of the multicellular constructs from the microfluidic device to another environment. For example, geometric cues at length scales of hundreds of micrometers can significantly affect cell migration, growth kinetics, and cancer invasion. This merits the development of strategies for the initiation and growth of 3D cancer organoids with various shapes, as well as for their extraction to an unconstrained environment.

[0009] A known technique of cell culturing is to form multicellular aggregates on a microfluidics device with a number of isolated microwells. Dissociated cell material is manually deposited through the top of each microwell at a time and the cell material in each microwell is subsequently covered with a drop of pre-polymerized hydrogel to allow cells to grow on the hydrogel and form hollow organoids.

[0010] Another known technique uses a microfluidics device with microwells interconnected by a fluid channel through side or bottom openings of the microwells. The microwells and the fluid channel are in the same substrate and different solutions are fed to the interconnected microwells through the same fluid channel. A hydrogel spheroid containing cell aggregates is formed within and trapped in each microwell. The cultured cells may be extracted through the fluid channel after the hydrogel is dissolved.

[0011] In conventional technologies for extracting the cells, the spheroid must be broken up and the cell’s attachment to the framework must also be taken apart. For example, in some conventional technologies, the gel may be broken by triggering enzymatic digestion of the gel, such as by using a collagenase, wherein the gel solution will digest the collagen and release the cells. In another example, a mechanical method may used to break the gel, such as by vortexting and vigorous pipetting to physically disrupt the gel and release the cells. It will be understood that to break the spheroids and turn them into cells, usually enzymatic digestion(e.g. using TrypLE, ACUTASE, or Typsin) and mechanical disruptions may be required.Example methods are discussed in L. D. Jager, Effect of enzymatic and mechanical methods of dissociation on neural progenitor cells derived from induced pluripotent stem cells, Advances in Medical Sciences, Volume 61 , 2016, 1 :78-84.

[0012] However, it has been recognized by the present inventors that the above known devices are not convenient to operate and use for providing a large number of organoids and tumoroids with different selected shapes. For example, it is difficult to control the shapes of the formed organoids and also be able to extract the formed organoids intact without dissolving the hydrogel from the microfluid device using these conventional technologies.

[0013] Thus, an improved microfluidics device and process are disclosed herein, which enable the convenient formation of any number of multicellular organoids or tumoroids of preselected shapes on the device, and selective extraction of individual organoids or tumoroids from the device. Specifically, isolated open molds for forming objects of different desired shapes are provided on a substrate and slidably coupled to a cover which includes one or more open fluid channels. The cover is slidably movable relative to the substrate so that different solutions can be selectively provided to the individual open molds through a selected channel on the cover at different times during formation of the objects, and to expose the open molds to allow convenient removal of the formed objects without breaking or damaging the formed objects. The formed objects include multicellular organoids or tumoroids. The individual open molds can be addressable so that the organoids or tumoroids can be selectively formed and removed based on their addresses.

[0014] According to an aspect of the present disclosure, there is provided a microfluidics device for forming multicellular constructs of selected shapes, comprising: a bottom layer comprising a plurality of isolated open molds of selected shapes each having a top opening and a hydrophobic surface; and a top layer comprising a fluid channel extending from an inlet to an outlet, the fluid channel comprising an open channel having a bottom opening, the top layer slidably coupled to the bottom layer and movable among a plurality of positions relative to the bottom layer, wherein the plurality of positions comprises: (i) a first position in which the bottom opening overlaps with the top opening for delivering a solution comprising cells and a hydrogel precursor to the top opening through the open channel, (ii) a second position in which the top opening is covered by the top layer and offset from the bottom opening to isolate a volume of the solution to be gelled in the open mold, (iii) a third position in which the open mold is covered by the top layer to allow the isolated volume of the solution to be gelled and the cells in the open mold to be cultured to form a multicellular construct of a shape conforming to the selectedshape of the open mold, and (iv) a fourth position in which the top opening is uncovered by the top layer to allow selective removal of the multicellular construct formed in the open mold having a selected shape from the open mold through the top opening.

[0015] In some implementations, the fluid channel may comprise interleaved basins and plateaus positioned and arranged such that, in the first position, the basins of the fluid channel and the isolated open molds overlap with the fluid channel form a continuous zig-zag fluid path through each one of the open molds overlap with the fluid channel, and in the third position, the basins and the open molds are offset and the basins are isolated from one another by the plateaus.

[0016] In some implementations, the open channel may be a first open channel, and the top layer may comprise a second open channel overlapping the top opening in the third position for delivering a culturing medium to the top opening, wherein the second open channel is optionally continuous.

[0017] In some implementations, the selected shapes of the open molds may be individually selected and comprise different shapes.

[0018] In some implementations, the plurality of open molds may be arranged in an addressable grid and are individually addressed.

[0019] In some implementations, the addressable grid may comprise a plurality of rows of molds, each row of molds being aligned to simultaneously overlap with the fluid channel in the first position and simultaneously offset from the fluid channel in the second position.

[0020] In some implementations, the plurality of open molds may comprise 24 to 96 open molds.

[0021] According to another aspect of the present disclosure, there is provided a method of operating the device disclosed herein, comprising: positioning the top layer in the first position and delivering the solution comprising the cells and hydrogel precursor to isolated open molds through the open channel to fill the open molds with the solution; sliding the top layer from the first position to the second position, thereby isolating a volume of the solution to be gelled in each one of the open molds; sliding the top layer from the second position to the third position, and forming multicellular constructs in the open molds in the third position.

[0022] In some implementations, the method may comprise applying a negative pressure at the outlet of the fluid channel to draw air out of the fluid channel and the open molds and to draw the solution into the fluid channel to fill the open molds.

[0023] In some implementations, the method may comprise delivering a culturing medium in contact with the cultured cells in the open molds.

[0024] In some implementations, the method further may comprise agitating the cell culturing medium to improve perfusion between the cell culturing medium and the cultured cells.

[0025] In some implementations, the method may further comprise, moving the top layer from the third position to the fourth position, and removing the multicellular constructs from the open molds in the fourth position.

[0026] In some implementations, removing the multicellular constructs may comprise applying a negative pressure to suck the multicellular constructs of the open molds.

[0027] In some implementations, the hydrogel precursor may comprise a precursor for forming a nanofibrillar hydrogel.

[0028] In some implementations, the cells in the solution may have a concentration of (1.2 to 2.0) x 105cells / pL.

[0029] In some implementations, the method may further comprise incubating the cells in the open molds of the device in an incubator.

[0030] In some implementations, the multicellular constructs may comprise solid shapes mimicking solid physiological tissues.

[0031] According to another aspect of the present disclosure, there is provided a method for extracting multicellular constructs formed in the open molds of the device disclosed herein, the method comprising: moving the top layer of the device to the fourth position to expose the top openings of the open molds and removing the multicellular constructs from the open molds.

[0032] In some implementations, removing the multicellular constructs may comprise applying a negative pressure to suck the multicellular constructs out of the open molds.

[0033] In some implementations, the negative pressure to suck the multicellular constructs may be applied by forward-revers pipetting.

[0034] In some implementations, removing the multicellular constructs from the open molds may comprise selectively removing multicellular constructs from selected open molds.

[0035] In some implementations, the open molds may have different addresses, and selectively removing may comprise removing multicellular constructs from the selected open molds based on selected addresses of the open molds.BRIEF DESCRIPTION OF DRAWINGS

[0036] In the figures, which illustrate example embodiments:

[0037] FIG. 1 is a block diagram of a microfluidics device, according to an embodiment;

[0038] FIG. 2 is a block diagram of a fluid channel in the microfluidics device of FIG. 1 ;

[0039] FIG. 3 is a block diagram of an isolated open mold in the microfluidics device of FIG. 1 ;

[0040] FIGs. 4A-4D are schematic diagrams of the microfluidics device of FIG. 1 in different operating positions;

[0041] FIG. 5A is a front view of a microfluidics device, according to another embodiment;

[0042] FIG. 5B is a right view of the microfluidics device of FIG. 5A;

[0043] FIG. 50 is top view of the microfluidics device of FIG. 5A;

[0044] FIGs. 6A-6D are schematic diagrams of the microfluidics device of FIGs. 5A-5C in different operating positions;

[0045] FIG. 7 is a block diagram of a microfluidics system incorporating the microfluidics device of FIGs. 5A-5C, according to an embodiment;

[0046] FIG. 8 is a schematic perspective cross-sectional view of an example microfluidics device, according to an embodiment, within an example microfluidics system;

[0047] FIGs. 9-10 are partial, enlarged front sectional-elevation views of the microfluidics device of FIG. 8;

[0048] FIGs. 11A-11B are plan views of a top layer and a bottom layer, respectively, in the microfluidics device of FIG. 8;

[0049] FIG. 12A is a plan view of a portion of the microfluidics device of FIG. 8 in a first position;

[0050] FIG. 12B is a right sectional elevation view of the portion of FIG. 12A in the first position;

[0051] FIG. 13A is a plan view of the portion of FIG. 12A in a second position;

[0052] FIG. 13B is a right sectional elevation view of the portion of FIG. 13A in the second position;

[0053] FIG. 14A is a plan view of the portion of FIG. 12A in a third position;

[0054] FIG. 14B is a right sectional elevation view of the portion of FIG. 14A in the third position;

[0055] FIG. 15A is another plan view of the portion of FIG. 12A in the third position;

[0056] FIG. 15B is another right sectional elevation view of the portion of FIG. 15A in the fourth position;

[0057] FIGs. 15C-15D are perspective views of the microfluidics device of FIG. 8 placed on a rocker device;

[0058] FIG. 16A is a plan view of the portion of FIG. 12A in a fourth position;

[0059] FIG. 16B is a right sectional elevation view of the portion of FIG. 16A in the fourth position;

[0060] FIGs. 17A-17B are perspective views of the microfluidics system of FIG. 8 using a coupling mechanism;

[0061] FIG. 18 is a flow diagram illustrating a method for operating a microfluidics device, according to an embodiment of the present disclosure;

[0062] FIG. 19 depicts example shapes of mold opening shapes, microgels formed in the molds, and tumoroids formed from the microgels.DETAILED DESCRIPTION

[0063] In overview, isolated open molds are used to form multicellular constructs, such as organoids or tumoroids, of selected shapes including a hydrogel and selected cells. To conveniently provide the needed solutions to a number of the open molds during formation of the hydrogel and growth of the cells, a separate cover comprising open fluid channels is slidably coupled to a substrate on which the open molds are provided. The cover is also moveable to a position to expose the open molds to allow convenient removal of the formed constructs through the top openings of the open molds. The individual open molds on the same substrate can be individually addressed.

[0064] A “multicellular construct” as used herein refers to a construct that includes (i) a hydrogel having a crosslinked network of molecules forming a three-dimensional (3D) framework and (ii) multiple cells of two or more types self-organized into a 3D structure attached to the hydrogel framework. The hydrogel can provide an extracellular matrix (ECM) for the supported cells to attach and grow. For example, a multicellular construct may be a multicellular tumoroid or organoid. A multicellular construct may also include a multicellular aggregate.

[0065] Conveniently, multicellular constructs can be formed using such a device in an efficient and user-friendly manner, such that multiple multicellular constructs may be formed at once in a single device. Further, it allows addressable extraction of the intact tumoroids from the device with the cells still attached to the ECM while keeping the cell-ECM interaction intact.

[0066] In a particular embodiment, a microfluidics device includes a top layer and a bottom layer, which are slidably coupled to one another. The bottom layer includes a plurality of isolated open molds of various shapes. Each open mold may have its own selected shape, which may be unique or may be the same as other open molds in the device. Each open mold also includes a top opening and a hydrophobic surface. The hydrophobic surface may prevent material, such as tumor cells, hydrogel and / or cell culture from adhering to the interior of theopen mold. As discussed below, the hydrophobic surface may allow a multicellular construct, such as a tumoroid, to be extracted intact from its respective open mold.

[0067] The top layer includes at least one fluid channel. Each fluid channel extends from an inlet to an outlet of the fluid channel, and includes an open channel having a bottom opening.

[0068] Since the top layer is slidably coupled to the bottom layer, the top layer may be moved amongst a plurality of positions relative to the bottom layer. In a first position, the bottom opening of the at least one fluid channel is aligned or overlaps with the top opening of the open molds. In this first position, a solution including tumor cells and a hydrogel precursor may be delivered to the top opening of each open mold through the open channel. This provides a user- friendly and efficient solution for depositing tumor cells within multiple open molds at once.

[0069] The top layer may be moved to a second position relative to the bottom layer, in which the top opening of each open mold is covered by the top layer. However, the bottom opening of the channel used to deposit the tumor cells in the first position is offset from the top openings. In the second position, a volume of solution may be isolated in each open mold by the top layer. Conveniently, this second position may also remove excess tumor cells still present in the fluid channel away from each open mold.

[0070] The top layer may also be moved to a third position relative to the bottom layer, in which each open mold is covered by the top layer to allow the isolated volume of the solution in each mold to be gelled. The tumor cells in each open mold may also be cultured to form a multicellular construct of a shape conforming to the shape of the open mold. A cell culture medium may also be delivered to each of the open molds using a channel formed in the top layer. The cell culture medium may perfuse into each of the open molds using the channel. For example, molecular movement at the interface between the multicellular construct, such as tumoroid cells, and the cell culture medium may allow nutrients to perfuse into the tumoroid, as well as allowing waste / discharge from the cells to perfuse out of the open mold. The channel may conveniently allow the cell culture medium to be delivered to and perfuse into multiple open molds at once.

[0071] The top layer may also be moved to a fourth position, in which the top openings of the open molds are uncovered by the top layer. The fourth position may allow selective removal of the multicellular construct formed in each open mold. For example, each open mold may be addressed and, if desired, selected for extraction. A multicellular construct, such as a tumoroid, may be extracted from the top opening of the selected open mold in the device, without needing to extract other tumoroids at the same time. The tumoroid may also be extracted intact, i.e. within its cell matrix. They hydrophobic surface in each open mold may prevent the tumoroidfrom adhering to the mold walls as it is removed. Since the tumoroid may have the shape of the selected open mold, and since (in some examples) not all open molds may have the same shape, it may be convenient to extract a single tumoroid at a time from the device. However, it may also be possible to extract more than one tumoroid at a time, such as from more than one selected mold in the device at a time.

[0072] It will be appreciated that a microfluidics device disclosed herein may address one or more problems or drawbacks in conventional microfluid devices for forming organoids, and in particular allow for the efficient and user-friendly growth of multicellular constructs, while also enabling the addressable extraction of one or more tumoroids from the device at a time. In conventional static culturing techniques, well-plate or hanging drops are used to feed the cells. However, this technique is cumbersome and difficult to work with. In conventional dynamic culturing techniques, the cells are in fluid communication with one another and so are fed together. This technique prevents individual control of the cells.

[0073] In addition, microfluidics device disclosed herein may be used to deliver active ingredients to the multicellular constructs or cell aggregates. In some examples, liposomes may be used as carriers to deliver active ingredients to the cell aggregates. In other examples, active ingredient delivery may use other carriers such as nanoparticles. The microfluidics device may be used for screening nano-delivery systems like liposomes. In some examples, delivery mechanism for media, active ingredients and nanoparticles may be based on gravity-driven flow.

[0074] FIG. 1 depicts a microfluidics device 100, according to an embodiment. Microfluidics device 100 includes a top layer 102 and a bottom layer 104. Top layer 102 is a flat surface, such as a sheet, and includes a top surface and a bottom surface. Similarly, bottom layer 104 is also a flat surface, such as a sheet, and includes a top surface and a bottom surface. Top layer 102 is disposed on bottom layer 104, such that the bottom surface of top layer 102 faces the top surface of bottom layer 104.

[0075] Top layer 102 and bottom layer 104 are slidably coupled with one another. Top 102 layer includes a coupling 103 and bottom layer 104 includes a coupling 105. Coupling 103 and coupling 105 interact to slidably couple top layer 102 to bottom layer 104. Coupling 103 and coupling 105 may include any conventional mechanisms for slidably coupling two flat surfaces together, including self-guiding tracks, a self-locking mechanism, keys, grooves and magnets.

[0076] One or more surfaces of top layer 102 and / or one or more surfaces of bottom layer 104 may have a low coefficient of friction. For example, the surfaces may be coated in a material with a low coefficient of friction. Top layer 102 and bottom layer 104 may be configuredto slide with respect to one another due to the one or more low coefficient of friction surface(s). It will be understood, however, that coupling 103 and coupling 105 may be used to prevent top layer 102 and bottom layer 104 from sliding at all times, such that top layer 102 and bottom layer 104 may be configured to maintain a particular position relative to one another.

[0077] Top layer 102 includes a fluid channel 106, which, as illustrated in FIG. 2, includes an inlet 112, an outlet 114 and an open channel 116.

[0078] Bottom layer 104 includes isolated open molds 108, each having a top opening 118 as illustrated in FIG. 3. Dimensions of top opening 118 may be between 100 to 2000 microns. Dimensions of top opening 118 may also be larger. As will be described in further detail below, top opening 118 may be large enough to accommodate gravity flow. Gravity flow may allow for better transmittal of nutrients, as will be discussed in further detail below.

[0079] As discussed in further detail below, top layer 102 is slidably moveable among a plurality of positions relative to bottom layer 104.

[0080] Fluid channel 106 is configured to receive material or a solution, which may be a liquid or aqueous substance. As depicted in FIG. 2, inlet 112 may be used for introducing a fluid material into fluid channel 106 and outlet 114 may be used for removing the fluid material from fluid channel 106. For example, the fluid material may be a solution containing a hydrogel precursor and a cell suspension or a solution containing a cell culture medium. In some examples, the fluid flow of the fluid material in fluid channel 106 may be driven by applying a suction or negative pressure at outlet 114. Suction or negative pressure at outlet 114 may also be used to draw air out of the fluid channel 106 and isolated open molds 108, to prevent air bubbles from forming within the fluid material.

[0081] Inlet 112 and outlet 114 may be interchangeable, such that inlet 112 may also be used as an outlet and outlet 114 may also be used as an inlet to reverse the direction of the fluid flow in fluid channel 106. For example, depending on the fluid material and the particular application, a positive pressure or negative pressure may be applied to inlet 112 or outlet 114.

[0082] Open channel 116 extends between inlet 112 and outlet 114 and the fluid material may flow from inlet 112 to outlet 114 through the open channel 116. Open channel 116 is open, i.e. , has a bottom opening used to deliver the fluid material into the isolated open molds 108 on the bottom layer 104. For this purpose, top layer 102 is slidably movable on the bottom layer 104 to align the bottom opening of the open channel 116 with the top openings 118 of isolated open molds 108, such that the bottom opening of channel 116 overlaps (or partially overlaps) with the top openings 118. In some embodiments, open channel 116 may be formed on the bottom surface of top layer 102, in the form of a continuous groove. In some embodiments, toplayer 102 may include more than one fluid channels or a fluid channel 106 may include more than one open channels. The bottom openings of the multiple open channels may form a repeating pattern. Different open channels may be fluidly isolated from one another, i.e. not in fluid communication with one another. When the bottom opening of the open channel 116 is aligned with the top openings of the open molds 108, the open molds 108 are in fluid communication with the open channel 116 and a fluid material may be delivered into the open molds 108 through open channel 116 or fluid channel 106.

[0083] Fluid channel 106 may include portions at or near the top surface of top layer 102, in addition to the open channel at the bottom surface of top layer 102. For example, fluid channel 106 may include fluid ports at the top surface of top layer 102 for receiving and removing fluid materials, which may include the inlet 112 and outlet 114.

[0084] In some examples, fluid channel 106 may be formed in a straight line in top layer 102. In other examples, fluid channel 106 may be formed in a zig-zag pattern within top layer 102 or upon a top or bottom surface of top layer 102. In further examples, fluid channel 106 may be formed in a curved line or other non-linear pattern across top layer 102, such as across bottom surface of top layer 102. Fluid channel 106 may additionally be formed in a straight, angled or zig-zag pattern through top layer 102, such as by varying the depth of penetration of fluid channel 106 through top layer 102.

[0085] Fluid channel 106 and open channel 116 may be microchannels. In some examples, the width and / or height of fluid channel 106 and open channel 116 may be from 10 to 100 pm. Other dimensions of fluid channel 106 and open channel 116 may also be in the order of tens of pm or less.

[0086] Top layer 102 may also include more than one fluid channel (not depicted). In these examples, the one or more channels may each be formed in different patterns and dimensions in top layer 102.

[0087] Bottom layer 104 may include a number of isolated open molds 108 arranged in a row so that the top openings 118 of the open molds 108 may be conveniently aligned with a single straight open channel 116 at the same time. In this way, open channel 116 overlaps (or partially overlaps) with the top openings 118 of the open molds 108. It will be understood that isolated open molds 108 may also be arranged in other configurations, such as in an inclined line, circular or zig-zag pattern.

[0088] The inner wall 102 of the open mold 108, such as the open mold 108a depicted in FIG. 3, has a surface that is hydrophobic and defines a mold volume of a selected shape for molding a hydrogel in the open mold into the corresponding shape. Open molds 108 are formedon the top surface of bottom layer 104. The shapes of the open molds 108 may be different or similar depending on the application. In some embodiments, an open mold 108a may have the shape of a microwell or cylindrical, but conveniently, different open molds may have shapes other than cylindrical as will be described further below.

[0089] A hydrophobic surface of the wall 120 can prevent the hydrogel solution and cells from adhering to the wall 120. Wall 120 may include one or more side walls and a bottom wall.

[0090] In some embodiments, the surfaces in the fluid channel 106 of top layer 102 may also be made hydrophobic.

[0091] A surface may be made hydrophobic by any suitable method known to those skilled in the art. For example, the wall may be formed of a hydrophobic material, or the surface may be treated or coated to render the surface hydrophobic.

[0092] Each open mold 108 provides a molding chamber. The molding chamber may be closed when the top opening 118 of the open mold is covered by a bottom surface of the top layer 102 in a selected position (see below), or may be open when the top opening is not covered by the top layer 102 and is exposed.

[0093] As noted earlier each open mold 108 may have a selected shape. For example, the top opening 118 of open mold 108a may have any desired profile. The profile of the top opening 118 may be rectangular, circular, square, rhombus, trapezoidal, oval, polygonal, T-shaped, 11- shaped, l-shaped, or irregular. Other shapes may also be possible. The profile and shape of an open mold may be selected to mimic a natural profile or shape of a physiological tissue or organ.

[0094] In some further examples, the three-dimensional shape of open mold 108a may also be selected, and may include a sphere, a disk, cylinder (oriented vertically or horizontally), rectangular prism, half-torus or some other shape. The three-dimensional shape of open mold 108a may be on the scale of millimetres. In other example, the three-dimensional shape of open mold 108a may be on a larger scale. Microfluidics device 100 provides significant flexibility in the dimensions of the three-dimensional shape of open mold 108a and other elements in microfluidics device 100.

[0095] It will be appreciated that the profile of the top opening 118 may be selected such that a multicellular construct, such as a tumoroid, formed within open mold 108 may be removed through top opening 118, i.e. the shape of top opening 118 is sized to permit a multicellular construct formed within open mold 108 to be removed.

[0096] Isolated open molds 108 are isolated from one another in the sense that the isolated open molds 108 are not in fluid communication with one another through any fluid conduit in thebottom layer 104, although it is possible to provide a fluid material to the mold chambers of different molds through the top openings 118 using the same open channel 116. Isolation during molding (gelation) and subsequent cell culturing allows the hydrogel formation and cell growth in different individual open molds 108 to be individually controlled and performed.

[0097] Device 100 is configured such that top layer 102 and bottom layer 104 can slidably move relative to one another among a plurality of different positions, as illustrated in the schematic diagrams of FIGs. 4A-4D. It will be understood that top layer 102 is depicted as partially transparent in FIGs. 4A-4D, such that bottom layer 104, which is disposed beneath top layer 102, may also be depicted. In a first position depicted in FIG. 4A, the bottom surface of top layer 102 faces the top surface of bottom layer 104, and the bottom opening of fluid channel 106 is aligned with or overlapping (partially or fully overlapping) at least one of the open molds 108.

[0098] In some examples, the first position may be used to deposit material or solution into top opening 118 of each of isolated open molds 108. In some further examples, the material or solution may be a cell suspension, which may include a hydrogel precursor, such as an EKGel precursor, and tumor cells. The cell concentration in the cell suspension may be, for example, between (1.2 to 2.0) x 105cells / pL. The first position may be used to deposit the cell suspension into top opening 118 of each of isolated open molds 108. Other materials may also be deposited into top opening 118 of each of isolated open molds 108.

[0099] EKGel is a nanofibrillar hydrogel that consists of Schiff base crosslinks between aide hyde groups on the surface of cellulose nanocrystals (a-CNCs) and amine groups of lysine residues in gelatin. See, for example, Prince, E., Cruickshank, J., Ba-Alawi, W. et al. Biomimetic hydrogel supports initiation and growth of patient-derived breast tumor organoids. Nat Commun 13, 1466 (2022). https: / / doi.org / 10.1038 / s41467-022-28788-6, incorporated by reference herein in its entirety.

[0100] The hydrogel precursor may also include a precursor for forming a nanofibrillar hydrogel, e.g. EKGel.

[0101] Multicellular aggregates formed with EKGel may have cell-cell junctions. EKGel undergoes crosslinking through a chemical reaction that initiates upon mixing its precursors. Throughout this process, cells remain suspended within EKGel. After the gelation, the cells start to attach to the scaffold and initiate the cell-cell and cell-ECM interactions. See Prince, E., Morozova, S., Chen and Z., Kumacheva. E. Nanocolloidal hydrogel mimics the structure and nonlinear mechanical properties of biological fibrous networks. Proc Natl Acad Sci USA. (2023) 120(51). https: / / doi.org / 10.1073 / pnas.2220755120, incorporated by reference herein in its entirety. See also Prince, E., Chen, Z., Khuu, N. and Kumacheva, E. Nanofibrillar HydrogelRecapitulates Changes Occurring in the Fibrotic Extracellular Matrix. Biomacromolecules 2021 22 (6), 2352-2362, DOI: 10.1021 / acs.biomac.0c01714, incorporated by reference herein in its entirety.

[0102] In some embodiments, in the first position, a bottom opening of open channel 116 of fluid channel 106 may be aligned or overlapping (partially or fully overlapping) with top opening 118 of each of isolated open molds 108, such as isolated open mold 108a, for delivering a solution including tumor cells and a hydrogel precursor to top opening 118 through open channel 116.

[0103] It will be appreciated that in the first position, isolated open molds 108 may be in fluid communication with one another through fluid channel 106, such that if material (e.g. a cell suspension) is deposited into inlet 112, the material may be deposited into top opening 118 of each of isolated open molds 108 connected by fluid channel 106. That is, top layer 102 may be configured to deposit a material (e.g. a cell suspension) into top opening 118 of each isolated open mold 108a.

[0104] It will further be appreciated that the pattern and dimensions of fluid channel 106 may be formed in top layer 102 to complement the pattern and dimensions of isolated open molds 108 formed in bottom layer 104 (and vice versa).

[0105] Top layer 102 may be configured to be transposed across bottom layer 104 from the first position to a second position, depicted in FIG. 4B. As used herein, transposing top layer 102 across bottom layer 104 may include physically moving top layer 102 while holding bottom layer 104 stationary, physically moving bottom layer 104 while holding top layer 102 stationary, or physically moving both top layer 102 and bottom layer 104.

[0106] Transposing top layer 102 to the second position may also include removing excess material trapped in fluid channel 106 away from isolated open molds 108. For example, excess cell suspension trapped in fluid channel 106 may be moved away from isolated open molds 108. The bottom surface of top layer 102 moving across the top surface of bottom layer 104 may also ensure that material contained in each of isolated open molds 108, such as cell suspension, may remain below top opening 118 within isolated open mold 108a.

[0107] Fluid channel 106 and isolated open molds 108 may be offset (i.e. no longer overlapping) in the second position such that, for each isolated open mold 108a, top layer 102 encloses top opening 118 to define a molding shape. The molding shape for each isolated open mold 108a may conform to the selected shape of isolated open mold 108a. In some examples, wherein a cell suspension was deposited into top opening 118 of each isolated open mold 108a while top layer 102 was disposed in the first position, the cell suspension may form into a cell-laden microgel or micro-volume in the molding shape. The cell-laden microgel may acquire the molding shape. The profile of the top opening 118 of each of isolated open molds 108 may be rectangular, circular, square, rhombus, trapezoidal, oval, polygonal, T-shaped, U-shaped, I- shaped, or irregular. Other shapes may also be possible.

[0108] It will be appreciated that in the second position, top opening 118 of each isolated open mold 108a is covered by top layer 102 and offset from the bottom opening of open channel 116 to isolate a volume of the solution to be gelled in isolated open mold 108a. The gelling processing will be discussed in more detail below.

[0109] Channel layer 102 may be further configured to be transposed across molding layer 106 from the second position (after fluid channel 106 has been flushed out) to a third position, depicted in FIG. 4C. The third position may align fluid channel 106 with isolated open molds 108, such that fluid channel 106 overlaps (partially or fully) with isolated open molds 108.

[0110] In the third position, each of isolated open molds 108, such as isolated open mold 108a, is covered by top layer 102 to allow the isolated volume of the solution, such as cell suspension, in isolated open mold 108a to be gelled. The third position may also allow the cells in the cell suspension, such as tumor cells, in isolated open mold 108a to be cultured to form an aggregate of cells, such as multicellular construct of a shape conforming to the selected shape (i.e. molding shape) of isolated open mold 108a.

[0111] In some embodiments, top layer 102 may be configured to deliver a material into top opening 118 of each isolated open mold 108a. In further examples, top layer 102 may be configured to deliver a cell culture medium into top opening 118 of each isolated open mold 108a. The cell culture medium may be for culturing an aggregate of cells from the cell-laden microgel in isolated open mold 108a. In some embodiments, the aggregate of cells may be a multicellular construct. The multicellular construct may conform to the selected shape of isolated open mold 108a, and in particular to the molding shape defined by top layer 102 enclosing top opening 118 in the second position.

[0112] In some embodiments, fluid channel 106 may be used to deliver the cell culture medium into top opening 118 of each isolated open mold 108a. In these embodiments, fluid channel 106 may be configured to be flushed out while top layer 102 is in the second position (or in some other position) to remove any excess cell suspension from fluid channel 106.

[0113] While top layer 102 is in the third position, the cell culture medium may be delivered in fluid channel 106, which may deliver the cell culture medium into top opening 118 of each isolated open mold 108a. It will be appreciated that in these embodiments, channel layer 102 may only include one fluid channel 106, although channel layer 102 may also include other fluidchannels.

[0114] Top layer 102 may also be configured to be transposed across bottom layer 104 to a fourth position, depicted in FIG. 4D. The fourth position may expose top opening 118 of at least one of isolated open molds 108, such as isolated open mold 108a, for extracting the aggregate of cells from within isolated open molds 108. Extracting the aggregate of cells intact may include extracting the aggregate of cells within its cell matrix. Since each isolated open mold 108a includes wall 120, which also includes a hydrophobic surface, the aggregate of cells and the cell matrix may not adhere to isolated open mold 108a while the aggregate of cells and the cell matrix are extracted.

[0115] While top layer 102 is disposed in the fourth position, top opening 118 of each isolated open molds 108, such as isolated open mold 108a, may be uncovered by top layer 102 to allow selective removal of the aggregate of cells (i.e. the multicellular construct) formed in isolated open mold 108a having a selected shape (i.e. conforming to the molding shape) from isolated open mold 108a through top opening 118.

[0116] In some embodiments, top layer 102 may include any number of fluid channels for different purposes.

[0117] Active ingredients may be delivered to isolated open molds 108 using fluid channel 106. Other fluid channels may not be required. However, other fluid channels may also be used to deliver active ingredients to isolated open molds 108, if desired. Active ingredient delivery may use carriers such as liposomes, nanoparticles, etc. For example, microfluidics device 100 may also be used for screening nano-delivery systems like liposomes. The delivery mechanism for media, active ingredients and nanoparticles may be based on gravity-driven flow.

[0118] For example, illustrated in FIGs. 5A-5C, a microfluidics device 200 may include a top layer 202, which includes fluid channel 206a and fluid channel 206b. Fluid channel 206a may be identical to fluid channel 106, such that fluid channel 206a may be used to deposit material (e.g. cell suspension) into top opening 118 of each of isolated open molds 108 in the first position of top layer 202. Fluid channel 206a also includes inlet 112 and outlet 114. In some examples, fluid channel 206b may be used to provide a cell culture medium to the open molds 108. Fluid channel 206b may be structured differently from fluid channel 106a Top layer 202 may otherwise be similar or identical to top layer 102, and microfluidics device 200 may be otherwise similar or identical to microfluidics device 100.

[0119] As well, top layer 202 of microfluidics device 200 includes a first open channel associated with fluid channel 206a and a second open channel associated with fluid channel 206b.

[0120] It will be appreciated that fluid channel 206a in top layer 202 may not always be aligned with or overlapping open mold 108 in bottom layer 104, as depicted in FIG. 5B and described in further detail below.

[0121] For example, device 200 is configured such that top layer 202 and bottom layer 104 can slidably move relative to one another among a plurality of different positions, as illustrated in the schematic diagrams of FIGs. 6A-6D. It will be understood that top layer 202 is depicted as partially transparent in FIGs. 6A-6D, such that bottom layer 104, which is disposed beneath top layer 202, may also be depicted.

[0122] As depicted in FIG. 6A, top layer 202 may be disposed in a first position on top of bottom layer 104, such that the bottom surface of top layer 202 may face the top surface of bottom layer 104. The first position may align or overlap (partially or completely) fluid channel 206a with isolated open molds 108. As discussed above with respect to device 100, in some examples, the first position may be used to deposit material into top opening 118 of each of isolated open molds 108. The material may be a cell suspension, although other materials may also be deposited into top opening 118 of each of isolated open molds 108.

[0123] In some embodiments, in the first position a bottom opening of open channel 116 of fluid channel 206 may be aligned or overlapping (partially or completely) with top opening 118 of each of isolated open molds 108, such as isolated open mold 108a, for delivering a solution including tumor cells and a hydrogel precursor to top opening 118 through open channel 116.

[0124] It will be appreciated that in the first position, isolated open molds 108 may be in fluid communication with one another through fluid channel 206a, such that if material (e.g. a cell suspension) is deposited into inlet 112 of fluid channel 206a, the material may be deposited into top opening 118 of each of isolated open molds 108 connected by fluid channel 206a. That is, top layer 202 may be configured to deposit a material (e.g. a cell suspension) into top opening 118 of each isolated open mold 108a.

[0125] It will further be appreciated that the pattern and dimensions of fluid channel 206a may be formed in top layer 202 to complement the pattern and dimensions of isolated open molds 108 formed in bottom layer 104 (and vice versa).

[0126] Top layer 202 may be configured to be transposed across bottom layer 104 from the first position to a second position, depicted in FIG. 6B.

[0127] It will be appreciated that in the second position, top opening 118 in each isolated open mold 108a is covered by top layer 202 and offset from the bottom opening of open channel 116 to isolate a volume of the solution to be gelled in isolated open mold 108a.

[0128] Top layer 202 may be configured to be transposed across bottom layer 104 from thesecond position to a third position, depicted in FIG. 60. Top layer 202 may be configured in the third position to align or overlap (partially or completely) fluid channel 206b with isolated open molds 108.

[0129] In examples where a cell suspension was deposited into top opening 118 of each isolated open mold 108a while top layer 202 was disposed in the first position, the cell suspension may form into a cell-laden microgel in the molding shape while top layer 202 is in the second position or while top layer 202 is in the third position. When top layer 202 is in the third position, the molding shape may be further defined by the height of fluid channel 206b. The cell-laden microgel may acquire the molding shape, defined in part by the height of fluid channel 206b.

[0130] In some embodiments, while top layer 202 is disposed in the third position, each of isolated open molds 108, such as isolated open mold 108a, is covered by top layer 202 to allow the isolated volume of the solution, such as cell suspension, in isolated open mold 108a to be gelled. The third position may also allow the cells in the cell suspension, such as tumor cells, in isolated open mold 108a to be cultured to form an aggregate of cells, such as multicellular construct of a shape conforming to the selected shape (i.e. molding shape) of isolated open mold 108a.

[0131] As noted above, top layer 202 may be configured to deliver a material or solution into top opening 118 of each isolated open mold 108a. In further examples, top layer 202 may be configured to deliver a cell culture medium into top opening 118 of each isolated open mold 108a. The cell culture medium may be for culturing an aggregate of cells from the cell-laden microgel in isolated open mold 108a. Fluid channel 206b may include an inlet (not depicted) for introducing the cell culture medium into fluid channel 206b and an outlet (not depicted) for removing the cell culture medium from fluid channel 206b.

[0132] It will be appreciated that since top layer 202 in microfluidics device 200 includes more than one fluid channel (in particular, fluid channel 206a and 206b), fluid channel 206a in microfluidics device 200 does not need to be flushed out to remove any excess cell suspension before cell culture medium may be delivered into fluid channel 206b.

[0133] Finally, top layer 202 may also be configured to be transposed across bottom layer 104 from the third position to a fourth position, depicted in FIG. 6D. In particular, top layer 202 may be configured to be transposed across bottom layer 104 from the second position to the fourth position. The fourth position may expose top opening 118 of at least one of isolated open molds 108, such as isolated open mold 108a, for extracting the aggregate of cells from within isolated open mold 108a. Extracting the aggregate of cells intact may include extracting theaggregate of cells within its cell matrix.

[0134] In some embodiments, while top layer 202 is disposed in the fourth position, top opening 118 of each isolated open molds 108, such as isolated open mold 108a, may be uncovered by top layer 202 to allow selective removal of the aggregate of cells (i.e. the multicellular construct) formed in isolated open mold 108a having a selected shape (i.e. conforming to the selected shape, which defines the molding shape) from isolated open mold 108a through top opening 118.

[0135] In some embodiments, isolated open molds 108 may be arranged on bottom layer 104 in an addressable grid. Isolated open molds 108 may also be individually addressable. For example, isolated open mold 108a may be individually addressable, such that isolated open mold 108a may be selected and the aggregate of cells contained within isolated open mold 108a may be extracted, even if the aggregate of cells in other isolated open molds 108 are not extracted. The addressable grid may include a plurality of rows of isolated open molds 108, each row of isolated open molds 108 being aligned to simultaneously overlap with the fluid channel 106. Material, such as a solution comprising cells and a hydrogel precursor or a culturing medium, may be delivered to one or more rows in the addressable grid at a time. It will be appreciated that delivering a material to one or more rows in microfluidics device 100 at a time may allow multiple experiments may be run in parallel.

[0136] Additionally, the selected shapes of isolated open molds 108 may be individually selected and include different shapes, such that isolated open mold 108a may have a first selected shape while another isolated open mold may have another selected shape.

[0137] Isolated open molds 108 may include tens or hundreds of open molds. In one particular example, isolated open molds 108 may include 24, 48 or 96 open molds. In the example of 96 open molds, the molds may be arranged, for example, in an array of 8 by 12 molds. In other examples, isolated open molds 108 may include several hundred open molds, which may also be arranged in an array. The number of molds and their configuration may depend on the particular application. For example, this may depend on the desired shape of the cells, whether different types of similar samples are desired, as well as other considerations. The microfluidics device described herein may provide a significant degree of flexibility.

[0138] As noted above, top layer 202 is a flat surface, such as a sheet. Bottom layer 104 is also a flat surface, such as a sheet. Top layer 202 includes coupling 203 and bottom layer includes coupling 105, such that top layer 202 and bottom layer 104 are slidably coupled with one another. Coupling 203 and coupling 105 may include any conventional mechanisms for slidably coupling two flat surfaces together, including self-guiding tracks, a self-lockingmechanism, keys, grooves and magnets.

[0139] However, in some alternate embodiments, top layer 202 and bottom layer 104 may be curved. In these embodiments, the curvature of top layer 202 and the curvature bottom layer 104 may complement one another, such that top layer 102 may slidably couple with bottom layer 104. In these alternate embodiments, coupling 203 and coupling 105 may include any conventional mechanism for slidably coupling two curved surfaces together.

[0140] As noted above, active ingredients may be delivered to isolated open molds 108 using culture channels. Other fluid channels may not be required. However, other culture channels may also be used to deliver active ingredients to isolated open molds 108, if desired. As noted above, active ingredient delivery may use carriers such as liposomes, nanoparticles, etc.

[0141] FIG. 7 depicts a microfluidics system 300, according to an embodiment. Microfluidics system 300 includes microfluidics device 200. It will be understood that in other embodiments, microfluidics system 300 may instead include microfluidics device 100. As discussed above, microfluidics device 200 includes top layer 202, while microfluidics device 100 includes top layer 102. Top layer 202 includes at least two fluid channels (i.e. fluid channel 206a and fluid channel 206b), whereas top layer 102 may only include a single fluid channel 106.

[0142] Microfluidics system 300 also includes a coupling mechanism 332, a deposition device 334 and an extraction device 336. Microfluidics system 300 is otherwise identical to microfluidics system 200. Microfluidics system may also include actuation device 333.

[0143] Coupling mechanism 332 may be used to control the slidable coupling between top layer 202 and bottom layer 104, in addition to coupling 203 and coupling 105. For example, coupling mechanism 332 may ensure that top layer 202 and bottom layer 104 are superimposed in the first position, the second position and the third position to a certain degree of accuracy, such as within 5 to 10% of the channel width. In some examples, the channel may be wider than the top opening of the open molds within this degree of accuracy, since a wider channel may allow for more media to fully perfuse the tissue contained within the open molds. Top layer 202 and bottom layer 104 may also be superimposed in the fourth position, in some examples. Coupling mechanism 332 may also be removable or adjustable to allow top layer 202 to slide relative to bottom layer 104. Coupling mechanism 332 may include any conventional means for coupling and / or controlling the coupling between top layer 202 and bottom layer 104, and may include a machine or device external to microfluidics device 200.

[0144] Coupling mechanism 332 may also include alignment mechanism and indicators (not shown) to ensure that top layer 202 and molding layer 102 are coupled in the right orientation,i.e. in the right position. Alignment indicators may include modified edges of top layer 202 and / or molding layer 102, notches or markings on or in top layer 202 and / or bottom layer 104, or tabs and grooves or other mechanical structures and markings to guide the sliding movement and alignment of top layer 202 relative to bottom layer 104. A combination of alignment mechanisms and indicators may be used.

[0145] Microfluidics system 300 may also include actuation mechanism 333. Actuation mechanism 333 may be used to transpose top layer 202 relative to bottom layer 104, such as by adjusting coupling 203 and coupling 105 or coupling mechanism 332. Actuation mechanism 333 may include conventional actuation mechanisms, such as servo actuators, linear actuators, motors, gears, electromagnets and other devices.

[0146] In some other examples, actuation mechanism 333 may be operated manually, such that top layer 202 may be transposed relative to bottom layer 104 manually, either by a person adjusting the layers relative to one another or manually with the assistance of the conventional actuation mechanisms discussed above.

[0147] In further examples, actuation mechanism 333 may be operated fully automatically, i.e. without significant human intervention.

[0148] Deposition device 334 may be used to deposit material into fluid channel 206a and / or fluid channel 206b. Deposition device 334 may include one or more types of devices for depositing material, such as cell suspension and cell culture medium. For example, deposition device 304 may include a pipette applied to inlet 112 of fluid channel 206a to apply material to fluid channel 206a, such as cell suspension when top layer 202 is in the first position. In some cases, another pipette or suction device may be applied to outlet 114 of fluid channel 206a to introduce suction at the other end of fluid channel 206a and help distribute the material, such as cell suspension, across fluid channel 206a.

[0149] Deposition device 334 may also include a pipette applied to the inlet of fluid channel 206b to apply material to fluid channel 206b, such as cell culture medium when top layer 202 is in second position 152 (or when to fluid channel 106 when top layer 102 is in the third position). In some cases, another pipette or suction device may be applied to the outlet of fluid channel 206b to introduce suction at the other end of fluid channel 206a and help distribute the material, such as cell suspension, across fluid channel 206b.

[0150] In some further examples, however, it may be desirable to perfuse the material, such as cell culture medium, throughout fluid channel 206b using gravity-driven flow. It may also be desirable to circulate the material back and forth throughout fluid channel 206b and isolated molding layers 108. In these examples, deposition device 334 may also include a rockerplatform to assist with gravity-mediated flow. Microfluidics device 200, including superimposed top layer 202 and molding device 104, may be placed on rocker platform. Rocker platform may tilt microfluidics device 200 at a pre-defined angle while material or solution, such as cell culture medium, is applied to inlet of fluid channel 206b. In some examples, suction force or a negative pressure may be applied to outlet of fluid channel 206b. Over time, the pre-defined tilt angle may change based on a pre-defined waveform, with a pre-defined frequency and amplitude (e.g. tilt angle).

[0151] In some specific examples, gravity-mediated flow may be achieved by placing a pipette tip filled with material, cell culture medium, at the inlet of fluid channel 206b. An empty pipette tip may be applied at the outlet of fluid channel 206b to collect cell culture medium pushed through fluid channel 206b by positive pressure generated by the full pipette tip at the input of fluid channel 206b.. In one example, 300 pL of cell culture medium, or more or less than 300 pL, may be applied at inlet 206b. Microfluidics device 200, including superimposed top layer 202 and molding device 104, may be placed on rocker platform before or after the pipette tip and / or suction are applied to the inlet and outlet. The rocker platform may be an Ohaus SHRK04DG, TEquipment, USA. The rocker platform may be set to rock at a 15° tilting angle and a tilting speed of 1 rpm (one full-rocking motion / min). As the material in the pipette tip applied to inlet is fully deposited into fluid channel 206b and the pipette tip becomes empty, the empty pipette tip at the inlet may be used to collect cell culture medium pushed towards it by positive pressure generated at the former outlet of fluid channel 206b. In this case, the formerly empty pipette tip applied at the outlet of fluid channel 206b may have collected a portion of material and so may be used to deposit material at the outlet of fluid channel 206b. It will be appreciated that the inlet and outlet of fluid channel 206b may thus be interchanged depending on what is applied at the inlet and outlet. As well, it will also be appreciated that rocker platform may allow material, such as cell culture medium, to constantly flow through fluid channel 206b, which may help remove waste products from cells within isolated open molds 108. If required, the waste products may be removed by removing the full pipette (whether it is at the inlet or outlet of fluid channel 206b) and applying another full pipette with fresh cell culture medium. Depending on the cell type, rocker platform may run in this way for two to four days, after which the cell-laden microgel in each isolated open mold 108a may be transformed into an aggregate of cells. The aggregate of cells may be in the molding shape defined by top layer 202 (including the height of fluid channel 206b) and the shape of isolated open mold 108a.

[0152] Extraction device 336 may be used to extract an element, such as an aggregate of cells, from isolated open mold 108a. Extraction device 336 may include, for example, a pipettefor extracting the element. Extraction device 336 may also include an array of pipettes for extracting elements from several or all of isolated open molds 108 at once. Other extraction techniques and devices may also be possible, such as using automated liquid handlers, mechanical micromanipulators or automated cell picking robots. For an example of using automated liquid handlers, see R. Cao et al., An Automation Workflow for High-Throughput Manufacturing and Analysis of Scaffold-Supported 3D Tissue Arrays. Adv. Healthcare Mater. 2023, 12, 2202422, incorporated by reference herein in its entirety. For an example of using micromanipulators and automated cell robots, see Z. Zhang et al., Robotic Micromanipulation: Fundamentals and Applications, Annu. Rev. Control Robot. Auton. Syst. 2019. 2:181-203, incorporated by reference herein in its entirety.

[0153] As noted above, top opening 118 of at least one of isolated open molds 108, such as isolated open mold 108a, may be exposed when top layer 202 is disposed in fourth position. The element, such as an aggregate of cells within isolated open molds 108 may be extracted from isolated open mold 108a by extraction device 306. Extracting the aggregate of cells intact may include extracting the aggregate of cells within its cell matrix. Since each isolated open mold 108a includes wall 120 with a hydrophobic coating, the aggregate of cells and the cell matrix may not adhere to isolated open mold 108a while extraction device 336 extracts the aggregate of cells and the cell matrix.

[0154] It will be appreciated that extraction device 336 (used while top layer 202 is disposed in fourth position 258) may allow a specific or selected isolated open mold 108a to be extracted without distributing other isolated open molds 108. The aggregate of cells (and cell matrix) in the selected isolated open mold 108a may be extracted without disturbing, extracting or damaging the aggregates of cells in other isolated open molds 108.

[0155] In some further embodiments, extraction device 306 may include other devices and techniques for extracting multiple aggregates of cells by breaking the cell matrix and collecting the cells in a solution.

[0156] It will be understood that in microfluidics system 300, microfluidics device 200 may be used interchangeably with microfluidics device 100, such that top layer 202 may be replaced with top layer 102 from microfluidics device 100.

[0157] FIG. 8 depicts a perspective cross-sectional view of an example microfluidics system 400, according to an embodiment. Microfluidics system 400 includes an example microfluidics device 401, according to an embodiment. Microfluidics device 401 includes a top layer 402 and a bottom layer 404.

[0158] Top layer 402 is a thin sheet with a top surface 430 and an opposite bottom surface432. Bottom layer 404 is also a thin sheet with a top surface 434 and an opposite bottom surface 436. Top layer 402 is in contact with bottom layer 404 such that bottom surface 432 of top layer 402 faces and is in contact with top surface 434 of bottom layer 404.

[0159] Top layer 402 is slidably coupled to bottom layer 404 and moveable among a plurality of positions relative to bottom layer 404.

[0160] Top layer 402 also includes a fluid channel 406a and bottom layer 404 also includes isolated open molds 408, similar to top layer 102 (and top layer 202) and bottom layer 104 discussed above. Although not depicted in FIG. 8, top layer 402 also includes at least one other channel.

[0161] Fluid channel 406a in top layer 402 includes an inlet 412a for introducing material into fluid channel 406a, an outlet 414a for removing the material from fluid channel 406a and an open channel 416a disposed between inlet 412a and outlet 414a for material to flow between inlet 412a and outlet 414a. Fluid channel 406a is formed in top surface 430 of top layer 402 and penetrates through top surface 430 to bottom surface 432 of top layer 402. In particular, inlet 412a and outlet 414a both penetrate through top surface 430 to bottom surface 432 of top layer 402. Open channel 416a is formed in bottom surface 432 of top layer 402. In the depicted example, open channel 416a is in the form of a groove in bottom surface 432. When top layer 402 is superimposed against bottom layer 404, as depicted, open channel 416a may form a closed path for material to flow, i.e. material may flow between the groove in bottom surface 432 of top layer 402 formed by open channel 416a and top surface 434 of bottom layer 404.

[0162] It will be appreciated that in other embodiments, open channel 416a may instead be formed between top surface 430 and 432 instead, such as in the form of a hollow tube, with periodic openings penetrating through bottom surface 432 for depositing material out of fluid channel 406a. In this embodiments, inlet 412a and outlet 414a may only penetrate from top surface 430 to open channel 416a, rather than all the way through to bottom surface 432.

[0163] Other configurations of open channel 416a and fluid channel 406a may also be possible.

[0164] In some examples, open channel 416a may connect inlet 412a to outlet 414a. Open channel 416a may be used to deposit material into isolated open molds 408 disposed beneath fluid channel 406a, when fluid channel 406a is aligned with or overlapping isolated open molds 408.

[0165] In some embodiments, fluid channel 406a or open channel 416a may include valleys419 and plateaus 420 and interleaved basins 421 , as depicted in FIG. 9 (also see FIG. 10). As illustrated in FIG. 9, which shows an enlarged view of a portion of a cross-section of fluidchannel 406a, the interleaved basins 421 and plateaus 420 are positioned and arranged such that, in the first position, the basins 420 and the isolated open molds 408a, 408b, which are aligned with the fluid channel 406a, form a continuous zig-zag fluid path through each of open molds 408a, 408b aligned with the fluid channel 406a. However, when the basins 421 and the open molds 408a, 408b are offset, i.e. , not overlapping, the basins 421 are isolated from one another by the plateaus 420. Thus, when the fluid channel 406a does not align and overlap with the open molds 408, fluid channel 406a and bottom layer 404 form a discontinuous fluid path, where the inlet 412a and outlet 414a are not in fluid communication.

[0166] Isolated open molds 408 include any number of isolated open molds represented by isolated open mold 408a and isolated open mold 408b for illustration purposes.

[0167] As discussed above, open channel 416a may include a repeating pattern of basins and plateaus, such that portions of open channel 416a are not in fluid communication with one another. Broken segments of open channel 416a (i.e. the zig-zag pattern) may also prevent inlet 412a and outlet 414a from being in fluid communication with one another. When fluid channel 406a is aligned or overlapping (partially or completely) with isolated open molds 408, as depicted, isolated open molds 408 may complement open channel 416a and connect the portions or broken segments of open channel 416a and isolated open molds 408 in fluid communication with one another. This may create a zig-zag pattern between fluid channel 406a and isolated open molds 408. For example, isolated open mold 408a may place portions of open channel 416a on either side of isolated open mold 408a in fluid communication with one another. As well, the broken segment or portion of open channel 416a between isolated open mold 408a and isolated open mold 408b may place isolated open mold 408a and isolated open mold 408b in fluid communication with one another (as well as the remaining isolated open molds 408). In addition, inlet 412a and outlet 414a may also be placed in fluid communication with each other using the configuration or zig-zag pattern described above.

[0168] Open channel 416a may also include bottom opening 417a, which aligns or overlaps with a top opening 118 of isolated open mold 408a to allow solution for delivering a solution from open channel 416a into isolated open mold 408a. Since open channel 416a may be broken, bottom opening 417a may include multiple bottom openings, which may align or overlap with top openings of other isolated open molds 408.

[0169] Microfluidics system 400 may also include a deposition device, which may include pipette tip 435 and suction device 437. Pipette tip 435 may include solution 440 for depositing into fluid channel 406a. As discussed above, the deposition device may also include other devices for depositing material into fluid channel 406a.

[0170] As depicted in FIGS. 8-9, pipette tip 435 deposits solution 440, which may be a cell suspension and / or a cell culture medium and / or some other material, into inlet 412a of fluid channel 406a. Solution 440 enters inlet 412a in top surface 430 of top layer 402 and penetrates through top layer 402 to bottom surface 432 of top layer 402 and exits into a first segment 416aa of open channel 416a of fluid channel 406a. Solution 440 passes through the closed space formed between the first segment 416aa of open channel 416a and top surface 434 of bottom layer 404 until solution 440 exits bottom opening 417a of open channel 416a and enters opening 418a of isolated open mold 408a. Solution 440 fills up isolated open mold 408a until it can continue to flow into a second segment 416ab of open channel 416a through opening 418a. Solution 440 then makes its way to an opening 418b of isolated open mold 408b, placing isolated open mold 408a and isolated open mold 408b in fluid communication with one another. This process may be repeated until all of inlet 412a and outlet 414b are in fluid communication with one another.

[0171] It will be appreciated that this zig-zag pattern may ensure that each of isolated open molds 408 is completely filled. In other embodiments where the open channel does not produce a zig-zag pattern, i.e. where the open channel runs unbroken and parallel above the isolated open molds 408, the solution deposited into the open channel may not fill all the isolated open molds 408. The solution instead travels the shortest path through the open channel, which may not include travelling through the isolated open molds 408.

[0172] FIG. 10 depicts a cross-section of the full fluid channel 406a, including inlet 412a and outlet 414a. As noted above, the zig-zag pattern or configuration described above may place inlet 412a and outlet 414a in fluid communication with each other. Solution deposited at inlet 412a (e.g. solution 440) may perfuse from inlet 412a, through the first segment 416aa of open channel 416a in fluid channel 406a, through isolated open mold 408a, through second segment 416ab of open channel 416a, through isolated open mold 408b, and onward through the next segments of open channel 416a and the next of isolated open molds 408 until the solution reaches outlet 414a. Outlet 414a may also apply a suction force or a negative pressure to draw, motivate or attract the solution (e.g. solution 440) to outlet 414a through open channel 416a and isolated open molds 408. Suction or negative pressure at outlet 414a may also be used to draw air out of open channel 416a and isolated open molds 108, to prevent air bubbles from accumulating within the solution and any tumoroids or gel that forms.

[0173] FIG. 11 A depicts a schematic bottom view of top layer 402, wherein the structures of bottom surface 432 of top layer 402 are visible. Similarly, FIG. 11B depicts a schematic top view of bottom layer 404, wherein the structures on top surface 434 of bottom layer 404 are visible.As described above, in microfluidics system 400, bottom surface 432 of top layer 402 may be in contact with the top surface of bottom layer 404.

[0174] In the depicted example, top layer 402 includes fluid channel 406a, as well as a fluid channel 406b, a channel 406c, a channel 406d, a channel 406e and a channel 406f. Fewer or more channels may also be possible. As discussed above, fluid channel 406a includes inlet 412a and outlet 414a, as well as open channel 416a. Open channel 416a may be broken into multiple segments, include first segment 416aa and second segment 416ab. Fluid channel 406b may also include an inlet 412b and an outlet 414b, as well as an open channel 416b. However, unlike open channel 416a in fluid channel 406a, open channel 416b in fluid channel 406b may not be broken into multiple segments, i.e. open channel 416b may only be one single segment always placing inlet 412b in fluid communication with outlet 414b. As a result, fluid channel 406a and fluid channel 406b may be formed differently from one another (i.e. patterned differently) in cover 402. In some other embodiments, fluid channel 406a and fluid channel 406b may be formed the same, e.g. fluid channel 406b may be broken into multiple segments or fluid channel 406a may not be broken into multiple segments. In further embodiments, other configurations and / or patterns of fluid channel 406a and fluid channel 406b may be possible.

[0175] In addition, channel 406c and channel 406e may be formed the same (i.e. patterned the same) as fluid channel 406a in top layer 402. Similarly, channel 406d and channel 406f may be formed the same (i.e. patterned the same) as fluid channel 406b in top layer 402. As such, channel 406c and channel 406e may be formed differently (i.e. patterned differently) from channel 406d and channel 406f in top layer 402.

[0176] Also in the depicted example, bottom layer 404 include isolated open molds 408. Isolated open molds 408 may have selected shapes or molding shapes. As used herein, selected shapes and molding shapes may be used interchangeable, although it will be understood that the selected shape of an isolated open mold may, in part, define the molding shape of that isolated open mold when top layer 202 encloses it.

[0177] Isolated open molds 408 may be disposed in one or more rows, such as mold row 442a, mold row 442b and mold row 442c. Mold row 442a may include isolated open mold 408a and isolated open mold 408a. Isolated open molds 408 may be identical, such that each of mold rows 442a, 442b, 442c are identical. In other examples (and as depicted in FIG. 11B), isolated open molds 408 may not all be the same, such that mold rows 442a, 442b, 442c are different from one another. For example, isolated open molds 408 in mold row 442a may be shaped differently than isolated open molds 408 in mold row 442b and isolated open molds 408 in mold row 442c. As depicted, the openings of isolated open molds 408 in mold row 442a may be anoval shape, while the openings of isolated open molds 408 in mold row 442b may be a rectangular shape and the openings of isolated open molds 408 in mold row 442c may be a T shape. In further examples, the openings of isolated open molds 408 may be any one of a circular shape, rectangular shape, oval shape, T shape, II shape and I shape. As well, the openings of isolated open molds 408 in mold row 442a may also have different shapes, such that, for example, isolated open mold 408a has a different shape than isolated open mold 408b. It will be appreciated that each of isolated open molds may also have a 3D shape defined by the opening of the mold.

[0178] In some further examples, each of mold rows 442a, 442b, 442c may contain the same or a different number of isolated open molds 408.

[0179] Isolated open molds 408 in each of mold rows 442a, 442b, 442c may complement channels 406a, 406c, 406e, respectively. In particular, when cavity layer 402 is superimposed on bottom layer 404 such that channels 406a, 406c, 406e are aligned or overlapping (partially or completely) with mold rows 442a, 442b, 442c, respectively, mold rows 442a, 442b, 442c may place the inlet and outlet of each of channels 406a, 406c, 406e, respectively, in fluid communication with one another. As discussed above and depicted in the cross-section of microfluidics system 400, mold rows 442a, 442b, 442c and each of channels 406a, 406c, 406e, respectively, may form a zig-zag pattern. With respect to fluid channel 406a, isolated open mold 408a may place first segment 416aa of open channel 416a in fluid communication with second segment 416ab, etc.

[0180] It will also be appreciated, however, that if cavity 402 is transposed relative to molding layer 402 such that channels 406b, 406d, 406f are aligned or overlapping (partially or completely) with mold rows 442a, 442b, 442c, respectively, channels 406b, 406d, 406f may place isolated open molds 408 in each of mold rows 442a, 442b, 442c in fluid communication with one another. In particular, material deposited at inlet 412b in fluid channel 406b may be deposited by open channel 416b in fluid channel 406b into each of isolated open molds 408 in mold row 442a, all the way until the material exits at outlet 414b. Similar situations may occur in channels 406d, 406f and mold rows 442b, 442c, respectively.

[0181] It will be appreciated that in some examples, channels 406a, 406b may be associated with isolated open molds 408 in mold row 442a, while channels 406c, 406d may be associated with isolated open molds 408 in mold row 442b and channels 406e, 406f may be associated with isolated open molds 408 in mold row 442c.

[0182] FIGs. 12A-12B depict a plan view and a right sectional elevation view, respectively, of a portion of top layer 402 disposed in a first position on top of bottom layer 404, focussingonly on fluid channel 406a, fluid channel 406b and mold row 442a. It will be appreciated that, in the particular example of microfluidics system 400, other channels 406c, 406d, 406e, 406f and mold rows 442b, 442c are also present in top layer 402 and bottom layer 404, although not depicted in FIG. 12A.

[0183] The first position aligns or overlaps fluid channel 406a with isolated open molds 408 in mold row 442a for depositing a cell suspension 440a into the opening of each of isolated open molds 408. Cell suspension 440a may be first deposited into inlet 412a of fluid channel 406a and permeate through open channel 416a of fluid channel 406a towards outlet 414a. As cell suspension 440a permeates or travels through open channel 416a of fluid channel 406a, each of isolated open molds 408 in mold row 442a, including isolated open mold 408a, may be filled with cell suspension 440a.

[0184] In some embodiments, the cell suspension may be a solution containing tumor cells and a hydrogel precursor.

[0185] Although not depicted, cell suspension 440a may be deposited using a deposition device, such as pipette tip 435 applied at inlet 412a. Pipette tip 435 may deposit cell suspension 440a into inlet 412a. In some further examples, suction device 437 may provide suction force or a negative pressure to draw or attract cell suspension 440a through channel 406, i.e. from inlet 412a, through open channel 416a and towards outlet 414a. Suction or negative pressure may also be used to draw air out of the fluid channel 406 and isolated open molds 408, to prevent air bubbles from forming within the tumor cells or gels that may form within isolated open molds 408.

[0186] As depicted in FIG. 12B, cell suspension 440a may totally fill isolated open mold 408a, passing through opening 418a of isolated open mold 408a and filling open channel 416a. Other isolated open molds 408 in mold row 442a may be similarly filled.

[0187] In some embodiments, in the first position, bottom opening 417a of open channel 416a of fluid channel 406a may be aligned with or overlapping top opening 418a of each of isolated open molds 408 in mold row 442a, such as isolated open mold 408a, for delivering a solution to top opening 418a through open channel 416a. The solution may include tumor cells and a hydrogel precursor.

[0188] FIGs. 13A-13B depict a plan view and a right sectional elevation view, respectively, of a portion of top layer 402 being transposed from the first position to a second position relative to bottom layer 404. Top layer 402 is transposed in a direction D1 relative to bottom layer 404, although it will be understood that top layer 402 is moved in direction D1 such that fluid channel 406b may be aligned with or overlapping mold row 442a. Excess cell suspension 440a trappedin fluid channel 406a is moved away from isolated open molds 408 in mold row 442a, whereas cell suspension 440a up to the opening of each of isolated open molds 408 is not removed. For example, cell suspension 440a may still fill isolated open mold 408a up to opening 418a, even after top layer 402 is transposed from the first position to the second position.

[0189] It will be appreciated that in the second position, top opening 418a of each isolated open mold 108a is covered by top layer 402 and offset from the bottom opening of open channel 416a to isolate a volume of the solution to be gelled in isolated open mold 408a.

[0190] The second position offsets fluid channel 406a with isolated open molds 408 in mold row 442a, such that fluid channel 406a and isolated open molds 408 are no longer overlapping. For each of isolated open molds 408 in mold row 442a, top layer 402 encloses the openings of the isolated open molds 408. For isolated open mold 408a, top layer 402 encloses opening 418a of isolated open mold 408a to define a molding shape 444a for cell suspension 440a in isolated open mold 408a to form into a cell-laden microgel (not depicted) after incubation or some other technique, discussed in further detail below. As discussed above, molding shape 444a for isolated open mold 408a conforms to the selected shape for isolated open mold 408a.

[0191] FIGs. 14A-14B depict a plan view and a right sectional elevation view, respectively, of a portion of top layer 402 disposed in a third position relative to bottom layer 404, focussing only on fluid channel 406a, fluid channel 406b and mold row 442a. The third position may fluid channel 406b with isolated open molds 408 in mold row 442a.

[0192] It will be appreciated that in the third position, cell suspension 418a may fill isolated open mold 408a (and other isolated open molds 408) up to opening 418a. Any remaining cell suspension 418a which was deposited into fluid channel 406a in the first position may have been removed while top layer 402 was transposed from the first position to the second position.

[0193] Gelling may be performed while top layer 402 is disposed in the third position. For example, in isolated open mold 408a, cell suspension 440a may form into a cell-laden microgel 460 while top layer 402 is disposed in the third position. For example, cell suspension 440a in isolated open mold 408a may form into cell-laden microgel 460. As described in further detail below, the cell-laden microgel may only be formed after incubation or some other technique.

[0194] As noted above, the second position offsets fluid channel 406a with isolated open molds 408 in mold row 442a. For each of isolated open molds 408 in mold row 442a, top layer 402 encloses the openings of the isolated open molds 408. For isolated open mold 408a, top layer 402 encloses opening 418a of isolated open mold 408a to define molding shape 444a for cell suspension 440a in isolated open mold 408a to form into a cell-laden microgel 460.

[0195] In some further embodiments, since fluid channel 406b may enclose isolated openmolds 408 in mold row 442a, such as isolated open mold 408a, the molding shape for each of isolated open molds 408 in mold row 442a may be further defined by a height 448b of second fluid channel 406b. In the example of isolated open mold 408a, molding shape 444a may be further defined by height 448b of second fluid channel 406b, in addition to top layer 402 enclosing opening 418a of isolated open mold 408a.

[0196] Cell-laden microgel 444b in isolated open mold 408a (as well as other isolated open molds 408) may conform to molding shape 444a. In some examples, cell-laden microgel 444b may only form after incubation of microfluidics device 401 for a period of time at a certain temperature, such as 2 hours at 37 °C to allow for gel formation in the microgels, i.e. the formation of cell-laden microgel 444b from cell suspension 440a. It will be appreciated that other methods for generating cell-laden microgel may also be possible, including different incubation temperatures, times and other techniques. For example, the hydrogel can be formed by any known techniques, such as those provided in W. Hu et al., Advances in crosslinking strategies of biomedical hydrogels, Biomater. Sci., 2019,7, 843-855, incorporated by reference herein in its entirety.

[0197] In other examples where a UV-crosslinked hydrogel is used, the incubation parameters are similar as described above, except the timing may be determined by the irradiation time (e.g. 15-90 seconds) instead of the 2 hours required for EKGel gelation. The mold may be opened or closed during this time.

[0198] While top layer 402 is disposed in the third position, a cell culture medium 450 may be delivered into or put in contact with the opening of each of isolated open molds 408 in mold row 442a. This may occur before or after cell-laden microgel 444b is formed, i.e. before gelling is performed. Cell culture medium 450 may be delivered after cell-laden microgel 444b is formed to prevent cell culture medium 450 from washing out the microgel. FIGs. 15A-15B depict another plan view and cross-sectional view, respectively, of top layer 402 disposed in the third position relative to bottom layer 404, focussing only on fluid channel 406a, fluid channel 406b and mold row 442a. Cell culture medium 450 is delivered by top layer 402 into the opening of each of isolated open molds 408 in mold row 442, such as into top opening 118a of isolated open mold 408a. In the depicted example, cell culture medium 450 may be delivered into fluid channel 406b, such as into inlet 412b of fluid channel 406b, and dispersed throughout open channel 416b of fluid channel 406b until cell culture medium 450 reaches outlet 414b.

[0199] In the third position, each of isolated open molds 408, such as isolated open mold 408a, is covered by top layer 402 to allow the isolated volume of the solution in isolated open mold 108a, such as cell suspension, to be gelled. The third position may also allow the cells inthe cell suspension, such as tumor cells, in isolated open mold 108a to be cultured to form an aggregate of cells 470, such as multicellular construct of a shape conforming to molding shape 444a of isolated open mold 408a.

[0200] A deposition device may be used to deposit cell culture medium 450 into top layer 402, such as into fluid channel 406b. The deposition device may include pipette tip 435, which was used to deposit cell suspension 440a into fluid channel 406a. Pipette tip 435 may contain cell culture medium 450. A different pipette (not depicted) may also be used to deposit cell suspension 440a, or some other deposition device may be used. As well, a collection device, such as collection device 437, may also be used to collect cell culture medium 450 after it has flowed across fluid channel 406b and into each of isolated open molds 408 in mold row 442a. Collection device 337 may be another pipette tip in addition to pipette tip 435. Another or different collection device may also be used, such as a device recirculating cell culture medium 450 to pipette tip 435. It will be understood that positive pressure from pipette tip 435 may motivate, push or urge cell culture medium 450 across fluid channel 406b and into each of isolated open molds 408 in mold row 442a, before collection device 337 collects cell culture medium 450.

[0201] In some examples, it may be desirable to perfuse cell culture medium 450 throughout fluid channel 406b using gravity-driven flow. It may also be desirable to circulate cell culture medium 450 back and forth throughout fluid channel 406b and isolated open molds 408 in mold row 442a. In these examples, the deposition device may include a rocker platform 480 (in addition to pipette tip 435 with cell culture medium 450 and collection device 437) to assist with gravity-mediated flow, as discussed above and depicted in FIG. 15C. Microfluidics device 401 , including superimposed top layer 402 and molding device 404, may be placed on rocker platform 480 and rocker platform 480 may tilt superimposed top layer 402 and molding device 404 at a tilt angle A1 while cell culture medium 450 is applied to inlet 112a of fluid channel 406b and while collection device 437 collects cell culture medium 450 at outlet 114b of fluid channel 406b. In some examples, only positive pressure may be applied, such as by pipette tip 435. In other examples, both positive pressure and negative pressure may be applied, such as by pipette tip 435 and collection device 437. Cell culture medium 450 may flow through fluid channel 406b in direction D2 from inlet 112b to outlet 114b of fluid channel 406b, based on tilt angle A1 of rocker platform 480 and, in some examples, from positive pressure generated by pipette tip 435.

[0202] In some examples, the tilt angle of rocker platform 480 may change over time based on a pre-defined waveform, with a pre-defined frequency and amplitude (e.g. tilt angle). Forexample, rocker platform 480 may slowly decrease the tilt angle until it is zero and further reduce the title angle until it is negative. FIG. 15D depicts rocket platform 480 with a tilt angle A2, which may be equal in magnitude but opposite in sign to tilt angle A1 , i.e. A1 = -A2. In other examples, tilt A2 may have a smaller or large magnitude than tilt angle A1. It will be appreciated that the tilt angle of rocker platform 480 may be time dependent, such that the waveform is sinusoidal and the amplitude of the peak of the waveform is tilt angle A1 and the amplitude of the trough of the waveform is tilt angle A2. Other waveforms and amplitudes may also be possible.

[0203] For example, tilt angle A1 may be equal to 15° and tilt angle A2 may be -15°. Rocker platform 480 may vary the tilt angle at a tilting speed of 1 rpm (one full-rocking motion / min). Culture medium 450 in the pipette tip 435 applied to inlet may be delivered into fluid channel 406b while rocker platform 480 is at tilt angle A1 (or closer to tilt angle A1 than tilt angle A2), as depicted in FIG. 15C. It will be appreciated that culture medium 450 in the pipette tip 435 may also be applied to inlet while tilt angle is about 0 degrees, before rocker platform 480 begins to rock or tilt towards tilt angle A1. Pipette tip 435 may become empty as rocker platform 480 approaches tilt angle A2. The empty pipette tip 435 at inlet 412a may be used collect cell culture medium 450, and so the pipette may instead be denoted as collection device 437 and the inlet may be denoted as outlet 414b, as depicted in FIG. 15D. Similarly, the former collection device 437 may be denoted as pipette tip 435 as it is now full with cell culture medium 450 after collecting cell culture medium 450 at outlet 414b that had flowed through fluid channel 406b, as depicted in FIG. 15D when rocker platform is close to or at tilt angle A2. Former collection device, now pipette tip 435, may also apply positive pressure to fluid channel 406b. In some further examples, collection device 437 may also apply negative pressure at outlet 414b in addition to or instead of positive pressure, to assist with gravity-driven flow. It will be appreciated that inlet 412b and outlet 414b of fluid channel 406b may be interchanged depending on what is applied at inlet 412b and outlet 414b. As well, it will also be appreciated that rocker platform 480 may allow other material or solution, such as cell culture medium 450, to constantly flow or circulate throughout fluid channel 406b, which may help to deliver the nutrients and subsequently remove waste products from cells within isolated open molds 108. If required, the waste products may be removed by removing pipette tip 435 (whether at the inlet or outlet of fluid channel 406b) or funneling the material collected by collection device 437 to some external depository. A new pipette with fresh cell culture medium may be applied afterward. Depending on the cell type, rocker platform 480 may run in this way for two to four days, after which cellladen microgel 450 in each isolated open mold 1408a may be transformed into aggregate ofcells 470.

[0204] Microfluidics device 401 may also be maintained at a controlled environment, including a controlled temperature and humidity, during this time. For example, the controlled environment may be within an incubator, with an incubator temperature of 37 °C and a constant CO2 level of 5%. The relative humidity within the incubator may be within the range of 90-95%.

[0205] Cell culture medium may be prepared using a variety of different methods. Some methods are described, for example, in H. Wang, et al., Growth of MCF-7 breast cancer cells and efficacy of anti-angiogenic agents in a hydroxyethyl chitosan / glycidyl methacrylate hydrogel, Cancer Cell Int 17, 55 (2017), incorporated by reference herein in its entirety. Methods are also described, for example, in S. M. Gross, et al., A multi-omic analysis of MCF10A cells provides a resource for integrative assessment of ligand-mediated molecular and phenotypic responses, Commun Biol 5, 1066 (2022), incorporated by reference herein in its entirety.

[0206] FIGs. 16A-16B show a portion of top layer 402 disposed in a fourth position relative to bottom layer 404, focussing only on fluid channel 406a, fluid channel 406b and mold row 442a. The fourth position may expose top opening 418a of at least one of isolated open molds 408, such as isolated open mold 408a, for extracting aggregate of cells 470 from within isolated open mold 408a. Extracting aggregate of cells 470 intact may include extracting aggregate of cells 470 within its cell matrix.

[0207] In some examples, while top layer 402 is disposed in the fourth position, top opening 118a of each isolated open mold 408, such as isolated open mold 408a, may be uncovered by top layer 202 to allow selective removal of aggregate of cells 470 formed in isolated open mold 408a through top opening 118. Aggregate of cells 470 may be a multicellular construct having a selected shape conforming to molding shape 444a of isolated open mold 408a.

[0208] An extraction device 436, such as a pipette, may be used to extract aggregate of cells 470. The pipette may perform reverse pipetting to extract aggregate of cells 470. After removing aggregate of cells 470, aggregate of cells 470 may be transferred to a chamber or well on another device for further processing, experimentation or safekeeping.

[0209] Extraction of aggregate of cells 470 may only occur, in some examples, after a 48- hour culture in isolated open molds 408. In other examples, extraction of aggregate of cells 470 may occur sooner or longer than 48 hours after applying cell culture medium 450 to isolated open molds 408.

[0210] FIG. 17A depicts a perspective view of microfluidics system 400, according to another example. Microfluidics system 400 includes microfluidics device 401, as discussed above, with top layer 402 disposed in the first position relative to bottom layer 404. In the firstposition, fluid channel 406b is aligned with or overlapping isolated open molds 408 in mold row 442b.

[0211] Top layer 402 is slidably coupled to bottom layer 404 using a coupling mechanism 452. Coupling mechanism 452 may include one or more conventional coupling mechanisms (not depicted) which superimpose top layer 402 to bottom layer 404 but also allow top layer 402 to be transposed relative to bottom layer 404 when desired. For example, coupling mechanism 452 may include linear actuators to precisely push top layer 402 and bottom layer 404 with a control velocity.

[0212] Coupling mechanism 452 also includes alignment mechanisms 464, 466 formed in bottom layer 404 and top layer 402, respectively. When alignment mechanisms 464, 466 line up, it is apparent that top layer 402 is disposed in the first position relative to bottom layer 404. For example, alignment features may be included on the device. In another example, alignment pins may be used to align the top and bottom layers. In a further example, the device may be pre-aligned and / or a control system may determine the device’s alignment based on alignment features and adjust the alignment accordingly. It will be appreciated that other alignment mechanisms may be also possible.

[0213] FIG. 17B depicts another perspective view of microfluidics system 400, according to an example. As discussed above, microfluidics system 400 includes microfluidics device 401, with top layer 402 disposed in the third position relative to bottom layer 404. In the third position, fluid channel 406b is no longer aligned with isolated open molds 408 in mold row 442b.

[0214] Alignment mechanisms 464, 466 may also be used to verify when top layer 402 is disposed in the third position relative to bottom layer 404. Other alignment mechanisms not depicted may also be used.

[0215] In other embodiments, coupling mechanism 452 may include additional or different aspects, which may replace or supplement alignment mechanisms 464, 466.

[0216] As discussed above, microfluidics device 401 includes top layer 402 and bottom layer 404. In one example, top layer 402 and bottom layer 404 may be formed of poly(methyl methacrylate) sheets. Top layer 402 and bottom layer 404 may be patterned using a 3-axis milling machine (PCNC770, Tormach, USA). Afterward, top layer 402 and bottom layer 404 may be cleaned by ultrasound sonication in Milli-Q grade deionized water (DI, 18.2 MQ cm resistivity) and sterilized by spraying 70% ethanol. Prior to assembly, a hydrophobic coating (Rain-X, ITW Global Brands, USA) may be applied to top layer 402 and bottom layer 404, and top layer 402 and bottom layer 404 may be dried at 65 °C for 5 min, or at some other temperature for another duration of time. Subsequently, for sterilization, top layer 402 and bottom layer 404 may beexposed to UV irradiation (254 nm, 345 W cm-2) for 20 minutes, or less than or more than 20 minutes. Next, silicone oil (20 cSt, Sigma-Aldrich, USA) may applied to top layer 402 and bottom layer 404 to prevent possible leakage in microfluidics device 401 and to act as a lubricant during sliding of top layer 402 and bottom layer 404. Top layer 402 and bottom layer 404 may be superimposed under a stereoscope using the alignment-helping marks and coupling mechanism, and oil excess may be removed. Alignment features at the corners of the topographically patterned sheets (top layer 402 and bottom layer 404) may assist with assembly of microfluidics device 401.

[0217] FIG. 18 depicts a method S500 for operating a microfluidics device, such as microfluidics device 100, microfluidics device 200 or microfluidics device 401 in microfluidics system 400.

[0218] At S502, a gel solution is delivered to an open mold through an open channel to fill the open mold with the gel solution. The gel solution may include, for example, a solution including a hydrogel precursor and tumor cells.

[0219] For example, the top layer of the microfluidics device may be positioned at a first position to align the open channel with the top opening of the open molds, and a solution comprising tumor cells and the hydrogel precursor is delivered to the isolated open molds in the microfluidics device through the open channel to fill the open molds with the solution.

[0220] Using microfluidics device 401 as an example, top layer 402 may be positioned in the first position relative to bottom layer 404. The solution may be cell suspension 440a, which may include tumor cells and the hydrogel precursor. Cell suspension 440a may be delivered to isolated open molds 408 in microfluidics device 401. Cell suspension 440a may be delivered through, for example, open channel 416a in fluid channel 406a formed in top layer 402, as well as through other open channels in other fluid channels formed in top layer 402. Cell suspension 440a may be delivered through the open channels, such as open channel 416a, to fill isolated open molds 408 with cell suspension 440a.

[0221] The hydrogel precursor in the solution, such as cell suspension 440a, may include a precursor for forming a nanofibrillar hydrogel.

[0222] The tumor cells in the solution, such as cell suspension 440a, may have a concentration of (1.5 to 1.8) x 105cells / pL.

[0223] A negative pressure may be applied at the outlet of the fluid channel to draw the solution into the fluid channel to fill the open molds. For example, suction device 432b may be applied at outlet 414a of fluid channel 406a to draw cell suspension 440a to fill isolated open molds 408, such as isolated open molds 408 in mold row 442a. Negative pressure may alsodraw or remove air from the fluid channel and the open molds, to prevent air bubbles from accumulating in the cells or gel formed within the molds. In some examples, the flow rate in the fluid channel may be between 1.2 to 1.8 mL / hour.

[0224] At S504, the top layer is slid to cover the open mold and isolate a volume of the gel solution in the open mold.

[0225] For example, the top layer may be slid from the first position to a second position, thereby isolating a volume of the gel solution in each of the one of the open molds.

[0226] In the example of microfluidics device 401 , top layer 402 may slide or slip from the first position to the second position. A volume of cell-suspension may be isolated in each of isolated open molds 408. For example, a volume of cell-suspension 440a in isolated open mold 408a may be isolated to be gelled, i.e. to form into a cell-laden microgel 460. The slide or slip speed may be in the range of seconds.

[0227] Gelling may be performed using one or more of the methods described above, such as an incubation chamber. Gelling may also be performed at a later stage in method S500.

[0228] In some embodiments, for isolated open mold 408a, top layer 402 may enclose opening 418a of isolated open mold 408a to define a molding shape 444a for cell suspension 440a in isolated open mold 408a to form / gel into cell-laden microgel 460.

[0229] At S506, the gel solution is allowed to form into a hydrogel in the open mold and form multicellular constructs in the open mold.

[0230] For example, the top layer may be slid from the second position to a third position, and a multicellular construct may be formed in the open molds in the third position.

[0231] In the example of microfluidics device 401 , top layer 402 slides or slips from the second position to the third position. An aggregate of cells 470 is formed in each of isolated open molds 408, such as isolated open mold 408a, in the third position. Aggregate of cells 470 may be a multicellular construct. The slide or slip speed may be in the range of seconds.

[0232] In some embodiments, the cells in the microgel 450 may form aggregate of cells 470 after the cells in the open mold are exposed to a cell culture medium 450 for culturing the cells in the cell laden microgel 450 in each of isolated open molds 408.

[0233] The multicellular constructs, such as aggregate of cells 470, may include solid shapes mimicking solid physiological tissues.

[0234] At S504 or, in some embodiments, possibly at S506, a positive pressure may be applied at the inlet of the fluid channel (and / or a negative pressure may be applied at the outlet of the fluid channel) to draw a solution into the fluid channel to fill the open molds or provide thesolution to the top opening of the open molds. For example, collection device 437 may be applied at outlet 414b of fluid channel 406b to collect cell culture medium 450. Pipette tip 435 may create positive pressure to push, urge or motivate cell culture medium 450 across the channel to be delivered to isolated open molds 408, such as isolated open molds 408 in mold row 442a. In some further embodiments, collection device 437 may create negative pressure at the outlet.

[0235] At S508, the top layer of the microfluidics device may be moved to expose the top opening of the open mold. The multicellular construct may then be removed from the open mold.

[0236] For example, the top layer may be moved from the third position to a fourth position to expose the open molds, and multicellular constructs are removed from the open molds in the fourth position. In the example of microfluidics device 401 , top layer 402 may be moved to the fourth position to expose top openings of isolated open molds 408, such as top opening 418a of isolated open mold 408a. Aggregate of cells 470, which may be multicellular constructs, may be removed from each of isolated open molds 408, such as isolated open mold 408a.

[0237] In some examples, staining markers may be used to ensure the formation of mature tissue and indicate when the multicellular construct may be removed from the open mold.

[0238] Removing the multicellular constructs, such as aggregate of cells 470, may include applying a negative pressure to suck the multicellular constructs out of the open molds, such as one or more of isolated open molds 408. The negative pressure to suck the multicellular constructs may be applied by forward-reverse pipetting.

[0239] In addition, removing the multicellular constructs, such as aggregate of cells 470, from the open molds, such as isolated open molds 408, may also include selectively removing multicellular constructs from selected open molds, such as isolated open mold 408a.

[0240] In some examples, multicellular constructs may be removed using ultrasound waves. Microfluidics device 401 , or at least bottom layer 404, may be securely position. Ultrasound waves may be applied with a suitable prove or transducer. Careful adjustment of ultrasound intensity and positioning may be used to dislodge the tumoroids from the isolated open molds 408 without damaging the tumoroids.

[0241] The open molds, such as isolated open molds 408, may also have different addresses. For example, isolated open mold 408a depicted in FIG. 11 B may have address (1 , 1) and isolated open mold 408b may have address (1 , 2). In this example, the address may be (row, column). It will be appreciated that other addresses may also be possible. Selectively removing the multicellular constructs, such as aggregates of cells 470, may include removingmulticellular constructs from the selected open molds, such as isolated open mold 408a, based on selected addresses of the open molds, e.g. (1 , 1).

[0242] In addition, method S500, such as at S506, may include incubating the cells in the open molds of the device in an incubator. For example, cell-laden microgel 460 and / or aggregate of cells 470 within isolated open molds 408 may be incubated in an incubator (not depicted).

[0243] Method S500 may also include rocking the microfluidics device to rock a culturing medium in contact with the cultured cells in the open molds. For example, microfluidics device 401 may be placed on rocker platform 480 to rock cell culture medium 450 in contact with cellladen microgel 460 to form aggregate of cells 470 in isolated open molds 408. This rocking may redistribute the molecules in the channel to prevent build up of waste and achieve more efficient perfusion of the culturing medium within the cultured cells.

[0244] It will be appreciated that some of the operations at S502, S504, S506, S508 may be performed in a different order. As well, in some embodiments, different operations may be performed at different places, at different times, and by different users. For example, removal of the formed tumoroids may be performed separately by a different operator at a location remote from the location at which the tumoroids are formed.

[0245] FIG. 19 depicts example shapes of isolated open molds 408. As discussed above, cell-laden microgel 460 and then, after culturing with cell culture medium 450, aggregate of cells 470 may be formed in isolated open mold 408a. Cell-laden microgel 460 and aggregate of cells 470 may each acquire the shape of isolated open mold 408a.

[0246] Mold opening shapes 702 include various shapes for the profile of the top opening of each of isolated open molds 408, which include a circular shape 702a, rectangular shape 702b, oval shape 702c, T shape 702d, II shape 702e and I shape 702f, respectively. Other shapes may also be possible.

[0247] Microgel shapes 704 include the shape of cell-laden microgel 460 formed in an isolated open mold with circular shape 702a, rectangular shape 702b, oval shape 702c, T shape 702d, II shape 702e and I shape 702f, respectively. It will be appreciated that microgel shapes 704 correspond closely with mold opening shapes 702.

[0248] Tumoroid shapes 706 include the shape of aggregate of cells 470 formed in an isolating open mold with circular shape 702a, rectangular shape 702b, oval shape 702c, T shape 702d, II shape 702e and I shape 702f, respectively. It will be appreciated that tumoroid shapes 706 correspond closely with microgel shapes 704 and mold opening shapes 702.

[0249] Terms such as “above”, “below”, “top”, “bottom” and the like are used herein todescribe relative positions of components in reference to the orientation of components depicted in the figures, and not necessarily to the direction of gravity. Components may be used in different orientations. For example, a microfluidics device may be inverted so that the top layer is positioned beneath the bottom layer (with reference to the direction of gravity).

[0250] Of course, the above-described embodiments are intended to be illustrative only and in no way limiting. The described embodiments are susceptible to many modifications of form, arrangement of parts, details and order of operation. The invention is intended to encompass all such modification within its scope, as defined by the claims.

Claims

WHAT IS CLAIMED IS:1 . A microfluidic device for forming multicellular constructs of selected shapes, comprising: a bottom layer comprising a plurality of isolated open molds of selected shapes each having a top opening and a hydrophobic surface; and a top layer comprising a fluid channel extending from an inlet to an outlet, the fluid channel comprising an open channel having a bottom opening, the top layer slidably coupled to the bottom layer and movable among a plurality of positions relative to the bottom layer, wherein the plurality of positions comprises:(i) a first position in which the bottom opening overlaps with the top opening for delivering a solution comprising cells and a hydrogel precursor to the top opening through the open channel,(ii) a second position in which the top opening is covered by the top layer and offset from the bottom opening to isolate a volume of the solution to be gelled in the open mold,(iii) a third position in which the open mold is covered by the top layer to allow the isolated volume of the solution to be gelled and the cells in the open mold to be cultured to form a multicellular construct of a shape conforming to the selected shape of the open mold, and(iv) a fourth position in which the top opening is uncovered by the top layer to allow selective removal of the multicellular construct formed in the open mold having a selected shape from the open mold through the top opening.

2. The device of claim 1 , wherein the fluid channel comprise interleaved basins and plateaus positioned and arranged such that, in the first position, the basins of the fluid channel and the isolated open molds overlap with the fluid channel form a continuous zig-zag fluid path through each one of the open molds overlap with the fluid channel, and in the third position, the basins and the open molds are offset and the basins are isolated from one another by the plateaus.

3. The device of claim 1 or claim 2, wherein the open channel is a first open channel, and the top layer comprises a second open channel overlapping the top opening in the third position for delivering a culturing medium to the top opening, wherein the second open channel is optionally continuous.

4. The device of any one of claims 1 to 3, wherein the selected shapes of the open molds are individually selected and comprise different shapes.

5. The device of any one of claims 1 to 4, wherein the plurality of open molds are arranged in an addressable grid and are individually addressed.

6. The device of claim 5, wherein the addressable grid comprises a plurality of rows of molds, each row of molds being aligned to simultaneously overlap with the fluid channel in the first position and simultaneously offset from the fluid channel in the second position.

7. The device of any one of claims 1 to 6, wherein the plurality of open molds comprise 24 to 96 open molds.

8. A method of operating the device of any one of claims 1 to 7, comprising: positioning the top layer in the first position and delivering the solution comprising the cells and hydrogel precursor to isolated open molds through the open channel to fill the open molds with the solution; sliding the top layer from the first position to the second position, thereby isolating a volume of the solution to be gelled in each one of the open molds; sliding the top layer from the second position to the third position, and forming multicellular constructs in the open molds in the third position.

9. The method of claim 9, comprising applying a negative pressure at the outlet of the fluid channel to draw air out of the fluid channel and the open molds and to draw the solution into the fluid channel to fill the open molds.

10. The method of claim 8 or claim 9, comprising delivering a culturing medium in contact with the cultured cells in the open molds.

11. The method of claim 10, further comprising agitating the cell culturing medium to improve perfusion between the cell culturing medium and the cultured cells.

12. The method of any one of claims 8 to 11, further comprising moving the top layer from the third position to the fourth position, and removing the multicellular constructs from the open molds in the fourth position.

13. The method of claim 12, wherein removing the multicellular constructs comprises applying a negative pressure to suck the multicellular constructs of the open molds.

14. The method of any one of claims 8 to 13, wherein the hydrogel precursor comprises a precursor for forming a nanofibrillar hydrogel.

15. The method of any one of claims 8 to 14, wherein the cells in the solution have a concentration of (1.2 to 2.0) x 105cells / pL.

16. The method of any one of claims 8 to 15, further comprising incubating the cells in the open molds of the device in an incubator.

17. The method of any one of claims 8 to 16, wherein the multicellular constructs comprise solid shapes mimicking solid physiological tissues.

18. A method for extracting multicellular constructs formed in the open molds of the device of any one of claims 1-7, the method comprising: moving the top layer of the device to the fourth position to expose the top openings of the open molds and removing the multicellular constructs from the open molds.

19. The method of claim 18, wherein removing the multicellular constructs comprises applying a negative pressure to suck the multicellular constructs out of the open molds.

20. The method of claim 19, wherein the negative pressure to suck the multicellular constructs is applied by forward-revers pipetting.

21. The method of any one of claims 18 to 20, wherein removing the multicellular constructs from the open molds comprises selectively removing multicellular constructs from selected open molds.

2. The method of claim 21 , wherein the open molds have different addresses, and selectively removing comprises removing multicellular constructs from the selected open molds based on selected addresses of the open molds.

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