A method of assaying a cell culture model

Abstract

A method for assaying the effects of a test agent on a microtissue comprises providing an assay plate system comprising an assay plate with a row of wells and a fluid conduit providing fluid communication along the row of wells, in each well contains a microtissue, flowing a test fluid containing the test agent along the fluid conduit and across each well, and imaging each of the microtissues in the wells The microtissue is disposed within a biopolymer hydrogel such as collagen or hyaluronic acid and is assayed in-situ in the biopolymer hydrogel in the well of the plate. The test agent may be an immune cell and the microtissue may be a cancer microtissue. The method allows for cells in the test fluid to attach to the hydrogel matrix and migrate into the hydrogel to interact with the microtissues held there. Entrapping a microtissue in a hydrogel within a well also allows for flow conditions of the test fluid in the well that are more physiologically relevant.

Description

[0001] A method of assaying a cell culture model

[0002] Field of the Invention

[0003] The present invention relates to a method of assaying a cell culture model, especially a 3-D cell culture model such as an organoid or microtissue. Also contemplated is an assay plate system for assaying a cell culture model.

[0004] Background to the Invention

[0005] During the process of drug discovery, test drugs need to be tested on cells to determine how they work and the effects of the drug on the cells. To date, the pharmaceutical industry has relied primarily on animal models and human cell line cultures that bear little resemblance to normal or disease human tissue, resulting in only one in every ten drugs making it through clinical testing. This high failure rate in clinical trials of drugs that make it through pre-clinical testing adds greatly to the cost of drug development.

[0006] Microtissues or organoids are tiny, self-organized three-dimensional tissue cultures that are derived from stem cells and other cell types such as primary cells. Such cultures can be crafted to replicate much of the complexity of an organ, or to express selected aspects of it like producing only certain types of cells. Organoids / microtissues may be grown from a variety of precursor cell types including stem cells — cells that can divide indefinitely and produce different types of cells as part of their progeny. Scientists have learned how to create the right environment for the precursor cells so they can follow their own genetic instructions to self-organize, forming tiny structures that resemble miniature organs composed of many cell types. Organoids / microtissues can range in size from less than the width of a hair to 5 mm. Many scientists believe that microtissues have the capacity to be more accurate and physiologically relevant models than existing animal models and that using organoids in pre-clinical testing of drugs will reduce the failure rate of drugs that enter clinical trials. These models will allow drug and vaccine manufacturers to bring products to market in a more timely and efficient manner. There is therefore a need for a technology to facilitate high throughput organoid screening that reproduces physiological conditions (e.g., temperature, CO2 and nutrients), facilitates fluid flow to the organoids, and allows organoids to be imaged.

[0007] WO 2024 / 013301 describes an assay plate system suitable for assaying in real-time cell culture models, especially 3-D cell culture models such as organoids, microtissues and spheroids, generally in a high-throughput manner. The system comprises a disposable assay plate containing one or more fluidic conduits, each having at least two or three wells, a fluid inlet conduit and a fluid outlet conduit. The wells are formed in an upper surface of an assay plate and have transparent bases allowing well contents to be imaged, and the conduits are defined by the upper surface of the assay plate, and a gasket that overlies the plate to form conduits in the plate comprising rows or columns of wells. Growing microtissues in wells of a plate is challenging. In addition, bringing agents in a test fluid into contact with microtissues in wells of a plate is challenging at the microfluidic level, especially when the agent is a cell such as an immune cell.

[0008] WO2024 / 026566 describes an assay system comprising an insert dimensioned to fit inside a well of microtiter plate, the insert having a plurality of microwells and at least two fluid inlet conduits configured to deliver test fluids to the microwells. In one embodiment, the microwell may contain a microtissue entrapped within an extracellular protein matrix. This assay system is configured to incubate a test fluid with the microtissue in the microwell for a defined period of time, which does not recapitulate physiologically relevant conditions.

[0009] It is an objective of the invention to overcome at least one of the above-referenced problems. Summary of the Invention

[0010] The Applicant has addressed the problem of the prior art by growing a microtissue in a hydrogel in-situ in a well of an assay plate, and then assaying the effects of a test agent such as a cell on the microtissue by flowing the test fluid across the well with the microtissue entrapped in the gel in the well. When the test agent is a cell, this allows for cells in the test fluid to attach to the hydrogel matrix and migrate into the hydrogel to interact with the microtissues held there. Entrapping a microtissue in a hydrogel within a well also allows for flow conditions of the test fluid in the well that are more physiologically relevant.

[0011] In a first aspect, there is provided a method for assaying the effects of a test agent on a cell culture model such as a microtissue comprising: providing an assay plate with a row of wells in which each well contains a cell culture model; passing a test fluid containing the test agent along a fluid conduit in fluid communication with each well; and imaging each of the cell culture models in the wells, wherein the cell culture model is disposed within a hydrogel and assayed in-situ in the hydrogel in the well of the plate.

[0012] Also described is an assay plate system for assaying a cell culture model, the assay plate system comprising: an assay plate comprising an upper surface and at least one row of wells, each well having an open top formed in the upper surface, a sidewall, and a closed transparent base; and a gasket comprising a plate having a top surface, a bottom surface, and at least one elongated recesses formed in the bottom surface that together with the upper surface of the assay plate defines a fluidic conduit for the at least one row of wells when the upper surface of the assay plate and bottom surface of the gasket plate abut, characterised in that at least one of the wells contains a cell culture model disposed within a hydrogel.

[0013] Also described is an assay plate system for assaying a cell culture model, the assay plate system comprising: an assay plate comprising an upper surface and at least one row of wells, each well having an open top formed in the upper surface, a sidewall, and a closed transparent base; and a gasket comprising a plate having a top surface, a bottom surface, and at least one elongated recesses formed in the bottom surface that together with the upper surface of the assay plate defines a fluidic conduit for the at least one row of wells when the upper surface of the assay plate and bottom surface of the gasket plate abut, characterised in that each well has a volume of about 0.5 to about 60uL.

[0014] Also described is a method for assaying the effects of a test agent on a cell culture model that employs an assay plate system as described herein, in which the method comprises: growing a cell culture model in each well of the row of wells; passing a test fluid containing the test agent along a fluid conduit in fluid communication with each well in the row of wells; and imaging each of the cell culture models in the wells, wherein each cell culture model is grown and assayed in a hydrogel in-situ a well.

[0015] In any embodiment, the method comprises: providing a composition comprising a biopolymer gel forming agent and a precursor cell; gelling the composition to form a hydrogel containing a precursor cell; and growing a cell culture model from the precursor cell in the hydrogel.

[0016] In any embodiment, the growing step comprises passing a growth fluid containing a growth agent along a fluid conduit in fluid communication with each well.

[0017] In any embodiment, the gelling step comprises methods such as ionic crosslinking, thermal gelation, hydrogen bonding, covalent bond formation, photo crosslinking, enzymatic crosslinking and / or hybrid crosslinking.

[0018] In any embodiment, the growing / maturation / maintenance step is performed in a well of the assay plate.

[0019] In any embodiment, the growing step is performed in a vessel and the hydrogel containing the cell culture is transferred from the vessel to a well of the assay plate prior to the assaying step.

[0020] In any embodiment, the cell culture model contained within the hydrogel is prepared by adding a preformed cell culture model to the hydrogel in a liquid form, adding the preformed cell culture model in liquid hydrogel to the well, and polymerising the liquid hydrogel around the preformed cell culture model in the well.

[0021] In any embodiment, the cell culture model contained within the hydrogel is formed by mixing cell culture model precursor cells with a hydrogel in liquid form to form a cell suspension, centrifuging the cell suspension while liquid to form a pellet of precursor cells surrounded by hydrogel in liquid form, and polymerising the hydrogel around the pellet of precursor cells.

[0022] In any embodiment, the test agent is a cell.

[0023] In any embodiment, the cell is an immune cell.

[0024] In any embodiment, the immune cell is selected from a neutrophil, eosinophil, basophil, mast cell, monocyte, macrophage, dendritic cell, natural killer cell, and lymphocyte (B-cell or T-cell).

[0025] In any embodiment, the cell culture model is a microtissue.

[0026] In any embodiment, the microtissue can be composed of individual cells or a combination of cells from the following organs, heart, lung, kidney, liver, pancreas, Gl tract, corneal, brain, breast, ovarian, cervical, prostate, and nerve microtissue. This list is not exhaustive, microtissues can be derived from any organ or tissue type.

[0027] In any embodiment, the microtissue is a cancer microtissue.

[0028] In any embodiment, the microtissue is about 2 to about 21 days old, or preferably about 3 to about 14 days old, or ideally about 4 to about 7 days old.

[0029] In any embodiment, the microtissue has a dimension of about 50 to about 1000 microns, preferably about 450 to about 550 microns.

[0030] In any embodiment, the hydrogel comprises a biopolymer gel forming agent.

[0031] In any embodiment, the biopolymer comprises or is selected from alginate, agarose, chitosan, gelatin, pectin, carrageenan, hyaluronic acid, Xanthan gum, starch based gels, cellulose based gels, fibrin or methylcellulose gels. Some examples include Matrigel, Gel MA or collagen 1 gel In any embodiment, the hydrogel comprises 0.5% to 2% or about 1 % biopolymer gel forming agent (w / v).

[0032] In any embodiment, each well has a volume of about 0.5 pl to about 60 pl, about 1.0 pl to about 30 pl, about 10 pl to about 20 pl.

[0033] In any embodiment, each well has a height of 0.5 to 2 mm and / or a width of 0.5 to 2 mm.

[0034] In any embodiment, each well has a height of about 1 mm and / or a width of about 1 mm.

[0035] In any embodiment, the elongated recess comprises at least one projection configured to project into one of the wells when the upper surface of the assay plate and bottom surface of the gasket plate abut, wherein the projection is dimensioned to modify fluid flow conditions in the well.

[0036] In any embodiment, the projection and the well are dimensioned to provide a fluid flow path in the well all around the projection when the upper surface of the assay plate and bottom surface of the gasket plate abut.

[0037] In any embodiment, a fluid flow envelope defined between the projection and the sidewall and base of the well has a width of about 100-2000, 500 to 1500, 700 to 1000 or 800-800 microns.

[0038] In any embodiment, the projection is centred in the well when the upper surface of the assay plate and bottom surface of the gasket plate abut.

[0039] In any embodiment, the well has an upper part and a lower part, in which the upper part has a curved conical recess shape and the lower part has a concave rounded recess shape. In any embodiment, the projection has a proximal stem part and a distal head part, in which the distal head part is dimensioned to nest within the well in a spaced-apartfrom the sidewall and base of the well.

[0040] In any embodiment, the distal head part has a curved conical section and a distal rounded tip.

[0041] In any embodiment, the distal head part comprises a proximal flange section, wherein the system is configured such that when the when the assay plate and gasket abut, the proximal flange section is flush with and spaced-apart from the upper surface of the assay plate.

[0042] In any embodiment, a shape and curvature of the curved conical section and the distal rounded tip section match the curvature of the sidewall and base of the well.

[0043] In any embodiment, the projection is configured to reduce the effective volume of the well by at least 40%, 50% or 60% (v / v). The “effective volume” refers to the space in the well that can receive fluid when the assay plate and gasket plate are assembled.

[0044] In any embodiment, the assay plate comprise a grid array of the wells including at least three rows of wells, and the gasket plate a grid array of projections corresponding to the grid array of wells.

[0045] In any embodiment, the well has an upper part and a lower part, in which the upper part has a curved conical recess shape.

[0046] In any embodiment, the lower part has a concave rounded (ideally hemi-spherical) recess shape.

[0047] The assay plate system typically comprises a cover plate configured to abut the top part of the gasket upon assembly of the assay plate system.

[0048] In any embodiment, the gasket is formed from PTFE (polytetrafluoroethylene, also known as Teflon). The use of PTFE for the gasket has been found to be advantageous as it can be machined to allow for more accurate manufacture, it does not interact with or adsorb molecules in the plate, and it is a stiffer material that silicone allow the gasket to be more easily handled.

[0049] In any embodiment, the top wall of the recesses (which form the top wall of the conduits when the system is assembled) are gas permeable and ideally liquid impermeable. The top of the recess typically has a thickness of 1 to 2 mm, 1.4 to 1.8 mm, or about 1.6 mm. When the gasket is formed from PTFE, a wall thickness of about 1 .6 mm is sufficiently thin to allow gas diffusion through the wall.

[0050] In any embodiment, the sidewalls of the recesses (which form the sides of the conduits when the system is assembled) have a height of 1 to 3 mm, 1 .5 to 2.5 mm, or about 2.0 mm.

[0051] In any embodiment, the assay plate comprises: a fluid inlet conduit for each column of wells having a first fluid inlet aperture disposed on the upper surface at a first end of each column of wells; and a fluid outlet conduit for each column of wells having a first fluid outlet aperture disposed on the upper surface at a second end of each column of wells.

[0052] The provision of a fluid inlet and outlet conduits at each end of a column of wells allows fluid to be directed along the columns of wells (for example, when growing cells into organoids).

[0053] In any embodiment, the fluid inlet conduit for each row or column of wells comprises a second fluid inlet aperture disposed on a lower surface of the assay plate at the first end of each row or column of wells.

[0054] In any embodiment, the fluid outlet conduit for each row or column of wells comprise a second fluid outlet aperture disposed on a lower surface of the assay plate at the second end of each row or column of wells. The arrangement of the fluid inlet and outlet conduits allows assay fluids to be provided to the columns or rows of wells from underneath the plate.

[0055] In any embodiment, the assay plate system comprises a base plate comprising a central through aperture comprising a recessed shoulder that extends around a periphery of the central through aperture, in which the recessed shoulder is dimensioned to receive the assay plate. This provides an effective base for receiving the assay plate and gasket plate and retaining them in position during the assay.

[0056] In any embodiment, an upper surface of the base plate is configured for coupling to the cover plate such that when the base plate and cover plate are coupled together with the assay plate and gasket sandwiched between the base plate and cover plate, the cover plate abuts the top of the gasket plate.

[0057] In any embodiment, the recessed shoulder of the base plate is configured to allow the assay plate and gasket nest within the base plate.

[0058] In any embodiment, the assay plate comprises at least 2, 3, 4, 5, 6, 7 or 8 rows of wells.

[0059] In any embodiment, the assay plate comprises at least 2, 3, 4, 5, 6, 7 or 8 columns of wells.

[0060] Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.

[0061] Brief Description of the Figures

[0062] FIG. 1 is a perspective view of an assay plate forming part of one embodiment of an assay plate system of the invention.

[0063] FIG. 2 is a perspective view of an underside of a gasket plate forming part of one embodiment of an assay plate system of the invention. FIG. 3A is a side elevational view of an assembled assay plate system of the invention with the top surface of the assay plate of Figure 1 abutting the underside of the gasket plate of Figure 2.

[0064] FIG. 3B is a sectional view taken along the lines A-A of Figure 3A.

[0065] FIG. 3C is a detailed view of part of the sectional view of the assay plate system of Figure 3B.

[0066] Detailed Description of the Invention

[0067] All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

[0068] Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

[0069] Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0070] As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method / process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method / process steps. The cell culture model may be a 2-D or 3-D cell culture model. As used herein, the term “3-D cell culture model” refers to a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy. Examples include organoids, microtissues and spheroids. Organoids / microtissues are derived from one or a few cells from a tissue, embryonic stem cells or induced pluripotent stem cells, which can self-organize in three-dimensional culture owing to their self-renewal and differentiation capacities. Such 3-D cell culture models typically have a maximum dimension of 50-1500 pm. Examples of 3-D cell culture model include liver, hepatic, pancreatic, epithelial, kidney, cardiac, retinal, blastoid, glioblastoma, thyroid and testicular organoids.

[0071] As used herein, the term “3-D cell culture model precursor” or “precursor” refers to a cell or composition of cells capable of being cultured to form a 3-D cell culture model. The precursor may be a cell from a specific tissue, a cell line, a patient cell or tissue sample, an embryonic stem cell, or an induced pluripotent stem cell. The assay plate system of the invention may be used to assay cell culture models such as organoids and to grow 2-D or 3-D cell culture model from precursor cells. Growth generally comprises placing a suitable precursor cell type(s) into a well, adding assay fluid (which may be cell culture fluid containing agents to promote the growth of the desired 2-D or 3-D cell culture model), and recirculation of the assay fluid to the well or wells.

[0072] As used herein, the term “assay plate system” refers to an assay plate body having assay wells suitable for assaying a 2-D or 3-D cell culture model such as an organoid. The plate generally has at least two rows of wells each row comprising at least two or three assay wells and generally a fluid inlet conduit and fluid outlet conduit. The plate includes a gasket plate to form conduits containing rows or columns of wells providing fluidic connection between the fluid inlet conduit, the wells, and the fluid outlet conduit in series, for example a PTFE gasket) This allows an assay liquid to be supplied to the each well. The plate is generally planar and the wells are generally formed in the top of the plate. The wells of the assay plate body generally have a base formed of a light transparent material that allows the wells to be imaged with an imaging system disposed under the plate. The wells are generally U-bottom wells. The assay plate and spacer / gasket of the system of the invention are generally disposable, whereas the other parts of the system may be re-usable.

[0073] As used herein, the term “light transparent material” refers to a material that is transparent. The base of the wells is generally formed from a light transparent material allow the organoids / microtissue in the wells to be imaged with an imaging device from below the second plate. The light transparent material is generally a polymeric material suitable for melting and casting. The light transparent material may be PMMA, , cyclic olefin copolymer (COC), perfluoropolyether’s (PFPEs), polyurethane, Flexdym, polylactic acid (PLA), PDMS.

[0074] Exemplification

[0075] The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

[0076] Example 1 - Preparation of Microtissue in Hydrogel (Method I)

[0077] A 1 % collagen solution (w / v) is prepared. Before the collagen has polymerised, a preformed microtissue is added to the collagen solution. The preformed microtissue is about 300 pm in diameter and contains about 5000 cells. The gel and microtissue are then pipetted into a well of a plate. This is repeated for a number of wells on the assay plate. The plate is then placed in a fridge to allow the collagen solution to polymerise around the microtissue in the well. The well is a small well having a height of about 1 pm and a width at its widest point of about 1 pm.

[0078] Example 2 - Preparation of Microtissue in Hydrogel (Method II)

[0079] Microtissue precursor cells are added to a 1 % MATRIGEL solution to make a cell suspension of about 100-200 cells per pl. The suspension is then vortexed to form a pellet of cells in the MATRIGEL. Before the MATRIGEL has polymerised, the pellet of cells and MATRIGEL (about 5 pl) is added to a well of a plate. This is repeated for a number of wells on the assay plate. Depending on the type of cells employed, the well may contain a single microtissue or a number of microtissues. The plate is then placed in a fridge to allow the collagen solution polymerise around the microtissue(s) in the well. The well is a small well that holds approximately 10 pl of liquid when full. The well is then perfused with a microtissue growth solution to allow growth of microtissue(s) from the pellet of cells in the hydrogel in the well of the plate.

[0080] Example 3 - Assay

[0081] An assay plate with a 3x3 grid array of wells is provided. Lung cancer, prostate cancer and liver cancer microtissues (MT’s) are prepared according to literature techniques, and added to the wells within a hydrogel according to the method of Example 1 so that a first row of wells each contains a lung cancer MT in a hydrogel, a second row of wells each contains a prostate cancer MT in a hydrogel, and a third row of wells each contains a liver cancer MT in a hydrogel. A test fluid containing an immune cell as test agent is then passed along a conduit in fluid communication with each well to allow the immune cell enter the wells and come into contact with the hydrogel in each well. After a period of time, the microtissues in the wells are then individually imaged.

[0082] Example 4 - Assay Plate System

[0083] Referring to the Figures, an assay plate system of the suitable for use in the method of the invention is illustrated. The assay plate system comprises an assay plate 1 comprising a rectangular planar plate with an upper surface 2, lower surface 3, and a peripheral sidewall 4 that extends around the upper surface 2 of the plate to define a recess 5. The plate is machined from acrylic but may also be may also be made by injection moulding using polystyrene, polypropylene or cyclo-olefins. The upper surface 2 has thirty six wells 6 arranged as a 4x9 grid array of wells comprising nine rows of four wells each. A fluid inlet conduit 7 is disposed at a first end of each row of wells, and a fluid outlet conduit 8 is disposed at a second end of each row of wells. These conduits are fluid ically connected to a fluidic system for moving fluid across the wells during use. Referring to Figure 2, the gasket plate (or spacer plate) 10 comprises a rectangular planar plate having an underside 11 with nine elongated recesses 12. Each recess includes four projections 13 that are equally spaced-apart along the recess. The plate 10 is dimensioned to nest snugly within the recess 5 of the assay plate with each recess 12 overlying a row of four wells 6. Thus, when the gasket plate 10 nests within the recess 5 of the assay plate, the upper surface 2 of the assay plate 1 and the recesses 5 define nine conduits in the assay plate system, one conduit for each row of wells to allow movement of fluid across the wells in the row from the fluid inlet conduit to the fluid outlet conduit.

[0084] Figures 3A to 3C illustrate an assembled assay plate system of the invention, indicated generally by the reference numeral 20, with the gasket plate 10 nested snugly within the recess 5 of the assay plate 1 . Referring initially to Figure 3C, each well 6 of the assay plate 1 has a upper part 6A and a lower part 6B. The upper part 6A of the well has a curved conical recess shape with a width across the top of about 3.5 mm and a width across the bottom of about 2.5mm. The lower part 6B has a concave hemispherical recess shape with a radius of curvature of about 1mm. The well has a height of about 2.5mm. A surface of the well 6 may be coated with PVA or Pluronic.

[0085] The projections 13 of the gasket plate 10 are dimensioned such that when the gasket plate 10 nests within the recess 5 of the assay plate, the projections extend into a centre of the wells 6 of the assay plate 1 leaving a gap 14 for fluid flow between the projection and the wall of the well. A gap of about 500 to 2000 microns may be employed to modify the shape and fluid flow dynamics of the wells sufficiently to allow efficient perfusion of the wells with a test fluid without disturbing or moving a microtissue disposed within the well. The gap chosen depends on a number of variables, for example the dimensions of the wells, the type of microtissue being assayed, the type of test fluid, and the type of assay being performed. In the embodiment shown, each projection 13 has a cylindrical stem part 14 and a head part 15 having a proximal flange section 16, an intermediate curved conical section 17 and a distal rounded tip section 18. The intermediate curved conical section 17 and a distal rounded tip section 18 are dimensioned to match the curvature of the well and ensure a gap of about 850 microns between the projection and the well. In use, the assay plate system is assembled providing nine fluidic conduits, each fluidic conduit in fluid communication with four wells containing a heart microtissue (first well), a liver microtissue (second well), a kidney microtissue (third well), and a lung microtissue (fourth well). An assay fluid is then pumped through the fluidic conduits of the base plate and the fluid inlet conduits of the assay plate to perfuse the microtissues in the rows of wells. The projections in the wells modified the fluid flow dynamics inside the wells to reduce the risk of disturbing or dislodging the microtissues from the wells while ensuring that the microtissues are adequately perfused with test fluid. This fluid exits through the fluid outlet conduits of the assay plate and the fluidic conduits of the base plate and is not recirculated. The assay plate system is configured to provide a different fluid to each row of wells. For example, a first row of wells may be provided with a fluid containing a first concentration of a first test molecule, a second row of wells may be provided with a fluid containing a second concentration of the first test molecule, a third row of wells may be provided with a fluid containing a first concentration of a second test molecule, and a fourth row of wells may be provided with a fluid containing a second concentration of the second test molecule, This fluidic system is then closed, and a fluid recirculation system (described in WO 2024 / 013301) for the nine fluidic conduits is actuated to recirculate fluid across the rows of wells to assay the effect that metabolites produced by one microtissue type has on another microtissue type.

[0086] Equivalents

[0087] The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto. Reference Numerals

[0088] Assay plate 1

[0089] Upper surface 2 (of assay plate)

[0090] Lower surface 3 (of assay plate)

[0091] Peripheral sidewall 4 (of assay plate)

[0092] Recess 5

[0093] Wells 6

[0094] Upper part 6A (of well)

[0095] Lower part 6B (of well)

[0096] Fluid inlet conduit 7

[0097] Fluid outlet conduit 8

[0098] Gasket plate (or spacer plate) 10

[0099] Underside 11 (of gasket plate)

[0100] Elongated recesses 12

[0101] Projections 13

[0102] Assay plate system 20

[0103] Cylindrical stem part 14 (of projection)

[0104] Head part 15 (of projection)

[0105] Proximal flange section 16 (of head part)

[0106] Intermediate curved conical section 17 (of head part)

[0107] Distal rounded tip section 18 (of head part)

[0108] Gap 19

Claims

CLAIMS:1 . A method for assaying the effects of a test agent on a microtissue comprising: providing an assay plate system comprising an assay plate with a row of wells and a fluid conduit in fluid communication with each well, in which each well contains a microtissue; passing a test fluid containing the test agent along the fluid conduit; and imaging each of the microtissues in the wells, wherein the test agent is a cell and the microtissue is disposed within a hydrogel and assayed in-situ in the hydrogel in the well of the plate, characterised in that the method comprises flowing the test fluid containing the test agent across each well in the row of wells.

2. A method according to any preceding Claim, in which the hydrogel comprises a biopolymer gel forming agent.

3. A method according to any preceding Claim, in which the test agent is an immune cell.

4. A method according to Claim 3, in which the microtissue is a cancer microtissue.

5. A method according to any preceding Claim, in which 1-20 pl of the hydrogel is added to the well.

6. A method according to any preceding Claim, in which the well has a volume of about 0.5 pl to about 60 pl.

7. A method according to any preceding Claim, in which the microtissue has a diameter of 50-1000 pm.

8. A method according to any preceding Claim, in which the microtissue comprises 200 to 10,000 cells and is assayed at about 3 to 21 days old.

9. A method according to any preceding Claim, in which microtissue is grown in the hydrogel in the well.

10. A method according to any of Claims 1 to 9, in which the microtissue contained within the hydrogel is prepared by adding a preformed microtissue to the hydrogel in a liquid form, adding the preformed microtissue in liquid hydrogel to the well, and allowing the liquid hydrogel polymerise around the preformed microtissue in the well.

11. A method according to any of Claims 1 to 9, in which the microtissue contained within the hydrogel is formed by mixing microtissue precursor cells with a hydrogel in liquid form to form a cell suspension, centrifuging the cell suspension while liquid to form a pellet of precursor cells surrounded by hydrogel in liquid form, and allowing the hydrogel to polymerise around the pellet of precursor cells.

12. A method according to any preceding Claim, in which the hydrogel comprises 0.5% to 2% (w / v) biopolymer gel forming agent.

13. A method according to Claim 12, in which the biopolymer is selected from the group consisting of alginate, agarose, chitosan, gelatin, pectin, carrageenan, hyaluronic acid, Xanthan gum, starch, cellulose, fibrinmethycellulose Matrigel, GelMA and collagen (Type 1 ).

14. A method according to any preceding Claim, in which the assay plate comprises an upper surface and each well comprises an open top formed in the upper surface, a sidewall, and a closed transparent base, wherein the assay plate system comprises a gasket comprising a plate having a top surface, a bottom surface, and at least one elongated recesses formed in the bottom surface that together with the upper surface of the assay plate defines the fluidic conduit for the at least one row of wells when the upper surface of the assay plate and bottom surface of the gasket plate abut.

15. A method according to Claim 14, in which the elongated recess comprises at least one projection configured to project into one of the wells when the upper surface of the assay plate and bottom surface of the gasket plate abut, wherein the projection is dimensioned to modify fluid flow conditions in the well, in which the projection is configured to define a fluid flow envelope between the projection and the sidewall and base of the well having a width of about 500 to about 1500 pm.

16. A method comprising growing a microtissue in a hydrogel in-situ in a well of an assay plate, and assaying an effect of a cell on the microtissue by flowing a test fluid containing the cell across the well with the microtissue entrapped in the hydrogel in the well.

17. A method comprising growing a microtissue in a hydrogel in-situ in a well of an assay plate, and assaying an effect of an immune cell on the microtissue by flowing a test fluid containing the immune cell across the well with the microtissue entrapped in the hydrogel in the well.

18. An assay plate system for assaying a cell culture model, the assay plate system comprising: an assay plate comprising an upper surface, a fluid inlet conduit, at least one row of wells, and a fluid outlet conduit, in which each well has an open top formed in the upper surface, a sidewall, and a closed transparent base; and a gasket comprising a plate having a top surface, a bottom surface, and at least one elongated recesses formed in the bottom surface that together with the upper surface of the assay plate defines a fluidic conduit for the at least one row of wells when the upper surface of the assay plate and bottom surface of the gasket plate abut providing fluidic communication from the fluid inlet conduit along the row and wells to the fluidic outlet conduit, characterised in that at least one of the wells contains a hydrogel and a cell culture model entrapped in the hydrogel.

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