Devices and methods for the preparation of an array of reproducible and homogeneous organoids

Abstract

The present invention relates to devices and methods for preparing an array of reproducible and homogeneous organoids, kits comprising such devices and arrays of reproducible and homogeneous organoids obtained by the methods.

Description

[0001] DEVICES AND METHODS FOR THE PREPARATION OF AN ARRAY OF REPRODUCIBLE AND HOMOGENEOUS ORGANOIDS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of biology, and more particularly to devices and methods for preparing arrays of reproducible and homogeneous organoids suitable for high-throughput analysis.

[0004] BACKGROUND OF THE INVENTION

[0005] Organoids are microtissues that recapitulate the complex structural organization and funchons of tissues and organs. They typically grow as self-organizing and self-renewing rounded structures that maintain populations of stem cells and specialized cells with phenotypes similar to those found in vivo. However, preparing organoid is susceptible to produce large variation in the phenotypic characteristics of organoids, particularly in their shape, size, cell-lineage ratios and functional properties.

[0006] The team of Lutolf and colleagues developed a platform aiming to overcome these drawbacks. This platform has been described in several patent applications and articles.

[0007] Firstly, W02016 / 1033002 discloses a platform preferably made of hydrogel comprising cavities, each cavity containing a plurality of deep microwells having a curved bottom. The distance between the microwells is minimal such that cells fall into a microwell. In general, the organoids are located at the bottom of the microwells.

[0008] W02018 / 050862 discloses a method for producing organoids in a microwell platform made of a biofunctional hydrogel. The microwells are large, e.g., a diameter ranging from 10 pm to 5 mm and a height ranging from 10 pm to 6 mm, the microwells being deeper than wide. In the experimental section, microwells diameters are between 500 and 1,500 pm and heights are from 360 to 1,800 pm. The number of cells per microwell is controlled by the cell density at the time of seeding. In this device, the majority of the organoids are located at the bottom of the microwells. WO2018 / 091677 discloses complex 3D structures made of hydrogel for the preparation of organoids.

[0009] More recently, Bliton et al. (2020) report the system designed by the team of Lutolf and colleagues for culturing homogeneous organoids at scale. The microwell platform is made of a biofunctional hydrogel. Two sizes of microwells were studied: 400-pm diameter microwells and 800-pm diameter microwells. Seeding was by gravity and the number of cells per microwell was controlled by the cell density at the time of seeding. 10, 50, 100 or 200 cells per microwell were seeded. Aggregates starting with 10 cells or less showed poor survival. Organoids formed from 50 seeded cells were able to polarize properly but were unable to bud successfully under differentiation conditions. Only microwells seeded with average densities of 100 and 200 cells showed growth patterns that were similar to those of organoids that polarize and form buds under differentiation conditions. The authors also showed that the microwell size had a strong influence on organoid longevity: organoids cultured in 400-pm diameter microwells were maintained for 8 days before showing signs of organoid death, whereas organoids cultured in 800-pm diameter microwells persisted for 15 days before showing a similar decline in health. In summary, it was concluded that large wells of 800-pm diameter with a density of at least 100 cells are necessary for the production of organoids.

[0010] In general, the known devices for the preparation of organoids have microwells that are wide and deep enough to completely contain the organoids into the microwells. Accordingly, the organoids are located at the bottom of the wells. Even if the proposed solutions offer improvements, the described systems still have drawbacks. In particular, these microwells have been designed to have a single organoid per well but the initial number of cells is not controlled. Poor reproducibility is observed as a consequence of the lack of control over the initial number of cells. In addition, there is a poor orientation of each organoid and several planes of observations are necessary.

[0011] Therefore, there is still a strong need for organoid-culture systems to ensure reproducible development of organoids for the preparation of tools suitable as models for the pharmaceutical industry and regulatory agencies.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention relates to devices and methods for preparing a large number of homogeneous organoids that are suitable for high-throughput screening, diagnostics and other assays such as toxicity measurements. The homogeneity of the organoids is regulated by the number of initial cells seeded in the microwells. In contrast to the methods previously disclosed in the prior art, the number of initial cells is controlled only by the diameter of the microwells. This control ensures the same rhythm of growth for cell proliferation and of differentiation, leading to nearly identical organoids.

[0014] Reproducible shapes and cell numbers are secured by the right design of microwells to host a specific target number of cells, the relevant coating of microwells to allow firm anchorage of organoids, inter-distances between microwells to prevent fusion between organoids, and the relevant embedding of cells in hydrogel such as Matrigel.

[0015] In contrast to the device previously disclosed in the prior art and as shown in Figures 8a and b, the depth of the microwells is selected to accommodate only about one layer of seeded cells. Therefore, seeding of the microwell plate and controlling the number of seeded initial cells are easily carried out through a centrifugation step followed by washes.

[0016] The device of the present invention allows the preparation of a unique array of organoids that are anchored at their base in the microwell but protrude from the microwell with a reproducible organization (see, Figure 8b). The appropriate size of the cavity allows to guarantee homogeneous growth while controlling initial cell number and positions of each organoid in space and time. The inventors identified that a coating of the microwells with adhesion protein(s) or peptide(s), such as laminin, is required for controlling the number of initial cells in the microwells and for providing an appropriate anchorage of organoids. This perfect control allows a high throughput analysis because the organoids are exactly in the same plane.

[0017] The array of organoids is within a single plane, i.e. at a particular focus, all organoids are viewed within the same focal plane, thereby facilitating the reproducibility and the automatic analysis. In particular, it allows high content screening, lower number of organoids required to conclude for a positive result and lower costs.

[0018] Accordingly, the present invention relates to a device for preparing an array of organoids comprising a plate and a microfabricated substrate comprising an array of microwells designed for controlling number of initial cells seeded into the microwells, wherein the microwells are defined by a depth, a length and a width; the microwells have a depth of about one cell diameter of the initial cells; surface area of the microwells depends on the number of initial seeded cells, and optionally is equal or substantially equal to the number of initial seeded cells * surface of one initial cell; ratio of length and width of the microwells is less than 2, preferably about 1; the width or length of the microwells is greater than the depth; the distance between microwells is large enough that the organoids are not in contact with each other, the microwells are coated with a composition comprising adhesion protein(s) or peptide(s); and optionally, a subset of microwells is surrounded by walls to cover the subset of microwells with a hydrogel, especially a hydrogel derived from natural extracellular matrix (ECM).

[0019] In a particular aspect, the microwells are coated with adhesion protein(s) or peptide(s) comprising at least 50, 60, 70, 80 or 90% of laminin. Optional ly, the microwells depth is constant and within the range of 8 to 35 pm or of 10 to 30 pm or of 10 to 25 pm or of 10 to 20 pm or of 10 to 17 pm.

[0020] Optionally, the surface area of the microwells is within the range of 150 to 96,000 pm2, preferably from 150 to 10,000 pm2; and / or the shape of the microwells are selected from round, square, triangle, oval, diamond, rectangle, hexagon, and octagon.

[0021] Optionally, the device comprises an array of identical microwells or multiple arrays of identical microwells, each array having a microwell surface area adapted for seeding a different number of initial cells, and optionally at least 2, 3, 4, 8, 16, 32 cells or more, optionally less than 300 initial cells.

[0022] Optionally, the device comprises microwells further comprising the initial seeded cells in the microwells and optionally covered by the hydrogel, especially a hydrogel derived from natural extracellular matrix (ECM).

[0023] In a particular aspect, the device comprises microwells further comprising organoids, said organoids being anchored into the microwells and protruding from the microwells.

[0024] Optionally, the plate comprises an array of wells comprising one or several arrays of microwells as defined herein.

[0025] The present invention further relates to the use of a device as described herein for preparing an array of organoids.

[0026] The present invention also relates to a method for preparing an array of organoids, comprising: a) providing a device as described herein; b) contacting the device with initial cells to be seeded, the upper side of microwells being up; c) centrifugating so that cells are placed into the microwells; optionally repeating step c) at least once or twice; d) washing to remove cells remaining outside the microwells; e) optionally adding a hydrogel, especially a hydrogel derived from natural extracellular matrix (ECM), to cover the seeded microwells so that the initial cells are embedded into the hydrogel; and f) placing the device in conditions suitable for growing the initial cells and forming organoids.

[0027] Optionally, the method comprises between steps c) and d) a step of incubating the device so that cells adhere to the microwells.

[0028] Optionally, the method comprises a preliminary step of determining the diameter of the initial cells, choosing a number of initial cells by microwell or a combination of numbers of initial cells by microwell and selecting a device having the appropriate size of microwells. Optionally, the method further comprises a step of observing the organoids, for instance by microscopy.

[0029] The present invention relates to an array of organoids obtainable or obtained by the method as described herein and to a kit for preparing an array of organoids comprising a device as described herein.

[0030] DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention relates to a device for preparing an array of organoids comprising a plate and a microfabricated substrate comprising an array of microwells designed for controlling number of initial cells seeded into the microwells, wherein the microwells are defined by a depth, a length and a width; the microwells have a depth of about one cell diameter of the initial cells; surface area of the microwells depends on the number of initial seeded cells and, optionally, is equal or substantially equal to the number of initial seeded cells * surface of one initial cell; ratio of length and width of the microwells is less than 2, preferably about 1; the width or length of the microwells is greater than the depth; the distance between microwells is large enough that the organoids are not in contact with each other, the microwells are coated with a composition comprising adhesion protein(s) or peptide(s) ; and optionally a subset of microwells is surrounded by walls to cover the subset of microwells with a hydrogel, especially a hydrogel derived from natural extracellular matrix (ECM).

[0032] The device comprises a plate and a microfabricated substrate comprising an array of microwells. The microwells are in the same plane. More specifically, the microwells are arrayed on a planar surface. In other words, at a particular focus, the microwells are all viewed within the same focal plane.

[0033] The size of the microwells is a key aspect of the invention because they have been designed by the inventors to ensure the control of the number of initial cells in the microwells.

[0034] The number of initial cells can be comprised between 1 and 500 cells, for instance between 2 and 300 initial cells. Optionally, the number of initial cells can be at least 2, 3, 4, 8, 16, 32 or more. Optionally, the number of initial cells can be 2, 3, 4, 8, 16, 32 or more. Optionally, the number of initial cells can be between 2 and 99. Optionally, the number of initial cells can be between 2 and 50 initial cells. The depth of the microwells is chosen to limit the number of layers of seeded cells, thereby improving the control of the number of initial cells. Indeed, this depth is important for allowing excess cells to easily go out of microwells during the washing steps. Accordingly, depth of the microwells is within the range of 1 to 2 cell diameters, preferably about 1 cell diameter. Especially, the depth of the microwells is selected to accommodate only one layer of seeded cells.

[0035] In a preferred aspect, the depth of the microwells is about 1 cell diameter. The cell diameter being often with the range of 10-20 pm, the depth of the microwells can be within the range of 8 to 35 pm or of 10 to 30 pm or of 10 to 25 pm or of 10 to 20 pm or of 10 to 17 pm. In a very specific aspect, the depth is within the range of 10 to 20 pm.

[0036] The inventors observed that, if the depth is too large (e.g., 80 pm), the device is not appropriate for the preparation of an array of organoids. In particular, cell apoptosis is observed if the depth of the microwells is too large (Fig 4d and e). On the opposite, if the depth of the microwells is too small, the trapping is poor (Fig 4a and b).

[0037] Preferably, width or length of the microwells is greater than the depth.

[0038] Optionally, the diameter of the initial cell can be determined as detailed in the example section. The diameter of the initial cell is its diameter in suspension (i.e., not the spread cell). In brief, the diameter the initial cell is measured after trypsinizing a monolayer of initial cells or any similar other means known by the person skilled in the art. Preferably, the diameter is determined in the middle planes of spherical cells. The diameter is preferably the mean of diameters measured on several initial cells. Alternatively, a distribution of diameters is measured and the diameter used in the design of the microwells is determined by selecting a point in this distribution, for instance based on the mean or median values of diameters.

[0039] All the microwells of a given array have the same depth or substantially the same. Optionally, when the support comprises multiple arrays, all of the microwells have the same depth. Alternatively, the arrays may have microwells of different depths from one array to another.

[0040] The microwells depth being selected, the number of initial cells has an impact on the surface area of microwells. Indeed, the surface area depends on the diameter of the initial cell seeded into the microwells and the number of initial cells to be seeded. More particularly, the surface area of the microwells is equal or substantially equal to the number of initial seeded cells * surface of one initial cell. By "substantially equal" to this product is meant between 0.5-fold and 5-fold, between 0.6-fold and 3-fold or between 0.7-fold and 2-fold, for example about 0.5-, about 0.6-, about 0.7-, about 0.8- , about 0.9-, about 1.1-, about 1.2-, about 1.3-, about 1.4-, about 1.5-, about 1.6-, about 1.7-, about 1.8-, about 1.9- or about 2-fold the number of initial seeded cells * surface of one initial cell. For instance, the surface area or diameter of the microwells can be selected according to the following table for initial cells having a diameter comprised between 10-20 pm.

[0041] Accordingly, a microwell adapted to host:

[0042] 2 cells has a surface area of 160-640 pm2and a diameter of 14-28 pm when the microwell shape is circular or substantially circular;

[0043] 4 cells has a surface area of 320-1,280 pm2and a diameter of 20-40 pm when the microwell shape is circular or substantially circular;

[0044] 8 cells has a surface area of 640-2,560 pm2and a diameter of 28-57 pm when the microwell shape is circular or substantially circular;

[0045] 16 cells has a surface area of 1,280 -5,120 pm2and a diameter of 40-80 pm when the microwell shape is circular or substantially circular;

[0046] 32 cells has a surface area of 2,560 -10,240 pm2and a diameter of 57-114 pm when the microwell shape is circular or substantially circular; and

[0047] 50 cells has a surface area of 4,000 -16,000 pm2and a diameter of 71-143 pm when the microwell shape is circular or substantially circular.

[0048] If the diameter of the initial cell is measured, then the number of cells seeded in the microwells can be closely defined and controlled. Inversely, if the device is standardized, then the person skilled in the art can determine precisely the number of initial cells by microwells.

[0049] The microwells may have any convenient shape. For instance, the microwells may have essentially a circular shape or a polygon shape. For instance, the shape can be round, square, triangle, oval, diamond, rectangle, hexagon, and octagon. In a particular aspect, the shape is circular. When the shape is circular or substantially circular, the size of the microwell (and the surface area thereof) can be defined by a diameter. When the shape a polygon, the size of the microwell (and the surface area thereof) can be defined by a width and a length.

[0050] The shape of microwells is not an elongated shape. Optionally, ratio of length and width of the microwells is less than 2, preferably about 1.

[0051] Optionally, the surface area of the microwells is within the range of 150 to 96,000 pm2, preferably from 150 to 10,000 pm2; and / or the shape of the microwells are selected from round, square, triangle, oval, diamond, rectangle, hexagon, and octagon.

[0052] Optionally, the microwell plate consists of microwells of identical diameter.

[0053] Alternatively, the microwell plate includes microwells of varying diameters. For instance, the microwell plates may comprise multiple arrays of microwells suitable for seeding different numbers of initial cells.

[0054] Optionally, the device comprises an array of identical microwells or multiple arrays of identical microwells, each array having a microwell surface area adapted for seeding a different number of initial cells, e.g., 2, 3, 4, 8, 16, 32 or more, optionally less than 300, less than 200 or less than 100 initial cells.

[0055] Optionally, the plate comprises an array of wells comprising one or several arrays of microwells as defined herein. The size of the well is suitable to contain said one or several arrays of microwells. More particularly, the plate may comprise an array of well which could be a 16, 96 or 384 well plate for cell culture. The array(s) of microwells are located at the bottom of some or each well of this 16, 96 or 384 well plate.

[0056] For instance, the microwells plate includes a combination of microwells arrays as detailed below: microwells suitable for seeding 2 cells, and / or microwells suitable for seeding 4 cells, and / or microwells suitable for seeding 8 cells, and / or microwells suitable for seeding 16 cells, and / or microwells suitable for seeding 32 cells.

[0057] More specifically, the microwell plate includes a combination of microwells arrays as detailed below: microwells with a surface area of 80-320 pm2and a diameter of 10-20 pm when the microwell shape is circular or substantially circular, and / or microwells with a surface area of 160-640 pm2and a diameter of 14-28 pm when the microwell shape is circular or substantially circular, and / or microwells a surface area of 320-1,280 pm2and a diameter of 20-40 pm when the microwell shape is circular or substantially circular, and / or microwells with a surface area of 640-2,560 pm2and a diameter of 28-57 pm when the microwell shape is circular or substantially circular, and / or microwells with a surface area of 1,280 -5,120 pm2and a diameter of 40-80 pm when the microwell shape is circular or substantially circular, and / or microwells with a surface area of 2,560 -10,240 pm2and a diameter of 57-114 pm when the microwell shape is circular or substantially circular, and / or microwells with a surface area of 4,000 -16,000 pm2and a diameter of 71-143 pm when the microwell shape is circular or substantially circular.

[0058] Optionally, the microwells plate includes a combination of 2, 3, 4, 5, 6 or more microwells arrays as defined above.

[0059] Optionally, the multiple arrays may have different shape of microwells. Optionally, the multiple arrays have a circular or substantially circular shape.

[0060] The distance between microwells is large enough that the organoids are not in contact with each other during their growth. For example, the distance between microwells is at least two, three, four or five times the width or length of the microwells. Optionally, the distance between microwells is at least two, three, four or five times the microwells diameter. In a particular aspect, the distance is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800 or 900 pm.

[0061] In a particular aspect, the microwell plate includes a microwells array or a combination of microwells arrays as detailed below: microwells with a depth of 10-20 pm, a surface area of 80-320 pm2and a diameter of 10- 20 pm when the microwell shape is circular or substantially circular, and a distance between microwells of at least 40, 60, 80 or 100 pm; and / or microwells with a depth of 10-20 pm, a surface area of 160-640 pm2and a diameter of 14- 28 pm when the microwell shape is circular or substantially circular, and a distance between microwells of at least 60, 80, 110 or 140 pm, and / or microwells with a depth of 10-20 pm, a surface area of 320-1,280 pm2and a diameter of 20-40 pm when the microwell shape is circular or substantially circular and a distance between microwells of at least 80, 120, 160 or 200 pm, and / or microwells with a depth of 10-20 pm, a surface area of 640-2,560 pm2and a diameter of 28-57 pm when the microwell shape is circular or substantially circular and a distance between microwells of at least 100, 150, 200 or 250 pm, and / or microwells with a depth of 10-20 pm, a surface area of 1,280 -5,120 pm2and a diameter of 40-80 pm when the microwell shape is circular or substantially circular and a distance between microwells of at least 160, 240, 320 or 400 pm, and / or microwells with a depth of 10-20 pm, a surface area of 2,560 -10,240 pm2and a diameter of 57-114 pm when the microwell shape is circular or substantially circular and a distance between microwells of at least 220, 330, 440 or 550 pm, and / or microwells with a depth of 10-20 pm, a surface area of 4,000 -16,000 pm2and a diameter of 71-143 pm when the microwell shape is circular or substantially circular and a distance between microwells of at least 300, 450, 600 or 750 pm.

[0062] Optionally, the density of microwells in an array or on the plate is at least one microwell per cm2, for example at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 microwells per cm2, preferably at least 200, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000 or 1,000,000 microwells per cm2.

[0063] The plate has to be convenient for confocal, optical and / or fluorescence microscopies. An appropriate plate can be any flat substrate promoting a good adhesion with the polymer used for the microstructure (i.e., the microfabricated substrate). In a more specific aspect, the plate is a plate of glass, preferably a silanised glass, more preferably a silanised glass coverslip. However, a quartz coverslip is also considered because it allows a high resolution. In a preferred aspect, the coverslip is as thin as possible. For instance, and without being restrictive, the thickness of 0.085 to 0.13 mm is convenient.

[0064] The microwell plate can be prepared by any suitable technique well-known by the person skilled in the art.

[0065] By "microfabricated substrate" is intended a microfabricated solid surface including, e.g., silicon, functionalized glass, germanium, ceramic, a semiconductor material, PTFE, carbon, polycarbonate, mica, mylar, plastic, quartz, polystyrene, gallium arsenide, gold, silver, metal, metal alloy, fabric, and combinations thereof. In particular, the microfabricated substrate is made of a solid material preferably biocompatible or with a surface treatment that makes it biocompatible. The following materials can be used for microfabrication: a polymer that can be crosslinked (PDMS, agar, polyacrylamide (PAM),...), a glassy material (polycarbonate (PC), polystyrene (PS)...), a metal or a semiconductor material.

[0066] Alternatively, the microfabricated substrate can be made of biofunctional hydrophilic synthetic hydrogel. For instance, the microfabricated substrate can be made of a macromolecule of hydrophilic polymers that are linear or branched, more preferably wherein the polymers are polyethylene glycolmolecules and most preferably wherein the polyethylene glycol molecules are selected from the group comprising: polyethylene glycol, polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, poly( ethylene oxide), polypropylene oxide, polyethylene glycol, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxy ethyl acrylate), poly(hydroxyethyl methacrylate) and mixtures thereof.

[0067] In a very specific aspect, the microfabricated substrate is not made of biofunctional hydrophilic synthetic hydrogel. In a very particular aspect, the microfabricated substrate is made of poly(dimethylsiloxane) (PDMS).

[0068] The plate is compatible with liquid handling, automated liquid handling, high throughput screening and / or micro-pipetting.

[0069] The microwells are coated with a composition comprising adhesion protein(s) or peptide(s), such as laminin. Indeed, the inventors surprisingly observed that the coating has an impact on the initial seeding and the cell spreading. More specifically, the concentration of laminin for coating microwells is comprised within 1 to 10 pg / ml, optionally within 3 and 7 pg / ml, for instance about 5 pg / ml.

[0070] Optionally, the microwell plate is coated with a composition comprising adhesion protein(s) or peptide(s), including the space between microwells. The inventors designed the device to remove the remaining cells by a washing step even when the plate is fully coated.

[0071] Alternatively, only the microwells are coated with a composition comprising adhesion protein(s) or peptide(s).

[0072] Optionally, the coating material can comprise different macromolecules, in particular molecules promoting cell attachment such as extracellular matrix molecules. Examples of such molecules include, without being restrictive, laminin, collagen, vitronectin, polylysine, synthetic or biomimetic molecules, synthetic peptides, carbohydrates and the like. The coating material may comprise cadherins. It should be understood that the coating material can be formed by a single molecule or a mixture of two or more molecules.

[0073] In a particular aspect, the coating material comprises laminin. In particular, the coating material can be a mixture comprising at least 50, 60, 70, 80 or 90% in weight of laminin.

[0074] In a particular aspect, the coating is carried out with a biomaterial comprising high content of laminin, e.g., at least 50, 60, 70, 80 or 90% in weight of laminin. For instance, Matrigel® comprises approximately 60% laminin, 30% collagen IV and 8 % entactin. Alternatively, the coating can be carried out with a mixture of laminin and Matrigel or Cultrex BME, thereby increasing the concentration of laminin.

[0075] Optionally, the bottom of the microwell is coated with laminin and the side of the microwells may have a different coating.

[0076] Optionally, the device further comprises barrier / wall surrounding an array of microwells or multiple arrays of microwells (see Figure Id). This barrier or wall is suitable to cover an array or multiple arrays of microwells with a hydrogel. This layer of hydrogel can be referred as an overlay.

[0077] The overlay or the hydrogel is made of naturally derived biomaterials such as polysaccharides, gelatinous proteins or extracellular components comprising the following or functional variants thereof: agarose; alginate; chitosan; dextran; gelatin; laminin; collagens; hyaluronan; fibrin, and mixtures thereof. Alternatively, the overlay of hydrogel may be made of Matrigel, Cultrex BME, Myogel and Cartigel, or a combination of Matrigel, Cultrex BME, Myogel and Cartigel and a naturally derived biomaterial or biomaterials. In a preferred aspect, the overlay or hydrogel is Matrigel or the like.

[0078] The overlay or hydrogel is made of biofunctional hydrophilic synthetic hydrogel. The biofunctional hydrophilic synthetic hydrogel may be a macromolecule of hydrophilic polymers that are linear or branched, more preferably the molecules are selected from the group comprising: polyethylene glycol, polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, poly(ethylene oxide), polypropylene oxide, polyethylene glycol, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxy ethyl acrylate), poly(hydroxyethyl methacrylate) and mixtures thereof and most preferably polyethylene glycol.

[0079] In a preferred aspect, the overlay or hydrogel is Matrigel or the like.

[0080] The present invention further relates to a device as disclosed herein comprising microwells further comprising the initial seeded cells in the microwells and optionally covered by the hydrogel, especially a hydrogel derived from natural extracellular matrix (ECM) such as Matrigel or Cultrex BME. In a preferred aspect, the initial seeded cells in the microwells are covered by an overlay of hydrogel, especially a hydrogel derived from natural extracellular matrix (ECM) such as Matrigel or Cultrex BME. The initial cells are embedded by the hydrogel.

[0081] The initial cell can be cells from a tissue (e.g., a differentiated cell), embryonic stem cells or induced pluripotent stem cells. Optionally, the initial cells can be a mixture of cells, for instance of 2, 3, 4 or 5 different cells. Preferably, at least one of the mixture of cells is an embryonic stem cell or induced pluripotent stem cell. For instance, the initial cells can be a mixture of pluripotent stem cell-derived tissue-specific progenitors or relevant tissue samples with endothelial cells and mesenchymal stem cells. Stem cells may be isolated from tissue, organoid fragments.

[0082] In addition, the present invention relates to a device as disclosed herein comprising microwells comprising organoids, said organoids being anchored into the microwells and protruding from the microwells. In a preferred aspect, the organoids are covered or embedded by an overlay of hydrogel, especially a hydrogel derived from natural extracellular matrix (ECM) such as Matrigel or Cultrex BME. The organoids have been grown in situ from the initial seeded cells.

[0083] Organoids, including cell spheroids or clusters, are cellular three-dimensional structures of stem cells, organ-specific, tissue-specific or disease-specific cell types that develop and self-organize (or self-pattern) through cell sorting and spatially restricted lineage commitment in a manner similar to the situation in vivo. An organoid therefore represents the native physiology of the cells and has a cellular composition (including remaining stem cells and / or specialized cell or tissue types at different stages of differentiation) and phenotype that emulate the native organ, tissue and / or diseased cells and tissue situation (e.g. cancer, cystic fibrosis, Inflammatory Bowel Disease). Normal and / or diseased cells (e.g. cancer cells) can be isolated from any tissues or any cellular structures such as organoids or cancer organoids (also called tumoroids). The cells from which an organoid is generated can grow and / or differentiate to form an organ-like or disease-like tissue (e.g. cancer, cystic fibrosis, Inflammatory Bowel Disease) exhibiting multiple cell types that self-organize to form a structure very similar to the organ (i.e. cell differentiation) or diseased tissue (e.g. multicellular heterogeneity of tumors) in vivo. Optionally, the organoid can be with or without lumen.

[0084] The organoids can be for instance cerebral organoids, cardiovascular organoids, hepatic organoids, gastrointestinal organoids, intestinal organoids, stomach or gastric organoids, lingual organoids, thyroid organoids, thymic organoids, testicular organoids, prostate organoids, pancreatic organoids, lung organoids, kidney organoids, endometrial organoids, retinal organoids, glioblastoma organoids, myelinoid organoids, skin organoids and blood-brain barrier organoids.

[0085] In a preferred aspect, an organoid exhibits the following properties: it has multiple organ-specific cell types; it is capable of recapitulating some specific function of the organ (e.g. contraction, neural activity, endocrine secretion, filtration, excretion); its cells are grouped together and spatially organized, similar to an organ.

[0086] The present invention further relates to a kit for preparing an array of organoids comprising a device as disclosed herein. Optionally, the kit may further comprise a culture medium, and tissue specific factors, nutrients and morphogens, which are preferably provided in a separated vessel. Optionally, the kit further comprises initial cells, especially stem cells and / or differentiated cells, which are preferably provided in a separated vessel such as a cryotube. Optionally, the kit comprises a device as disclosed herein; a medium, comprising tissue specific factors, nutrients and morphogens; and the initial cells. Optionally, the kit may further comprise a leaflet providing instructions for use the kit.

[0087] The present invention relates to a method for preparing an array of organoids comprising: a) providing a device as disclosed herein; b) contacting the device with initial cells to be seeded, the upper side of microwells being up; c) centrifugating so that cells are placed into the microwells; d) washing to remove cells remaining outside the microwells; e) optionally, adding a hydrogel, especially a hydrogel derived from natural extracellular matrix (ECM), to cover the seeded microwells so that the initial cells are embedded into the hydrogel; and f) placing the device in conditions suitable for growing the initial cells and forming organoids.

[0088] In a particular aspect, the method for preparing an array of organoid comprises a step e) wherein a hydrogel, especially a hydrogel derived from natural extracellular matrix (ECM), is added to cover the seeded microwells so that the initial cells are embedded into the hydrogel.

[0089] In another particular aspect, the washing step d) is followed by step f) without step e). In this aspect, the seeded microwells are not covered with hydrogel.

[0090] The method may comprise a preliminary step of determining the diameter of the initial cells, choosing a number of initial cells by microwells or a combination of numbers of initial cells by microwells and selecting a device having the appropriate size of microwells.

[0091] In the step b), the device may be placed in a container suitable for centrifugation with the top of the device (i.e., the side containing the microwells opening) oriented to receive the centrifuged cells. The cells are added in the container as a cell suspension. Optionally, cell concentration of the initial cells contacted with the device is between 106and 4*106per ml.

[0092] In a specific aspect of the step c), the centrifugation is carried out at 500-2,000 rpm for 1-10 minutes, for instance at 750-1,750 rpm for 2-5 minutes, more specifically at about 1,000-1,500 rpm for 3 minutes. Optionally, the centrifugation is carried out at 100-200g acceleration for 1-10 minutes. Optionally, the centrifugation can be carried out several times, for instance 2, 3 or 4 times, more preferably 2 or 3 times.

[0093] Between the step c) and d), the device can be incubated, in particular to allow initial cells to adhere to the microwells. The incubation can last for instance about 10 minutes. In a specific aspect of the step d), the wash step is carried out once or can be repeated.

[0094] Optionally, the steps c) and d) are repeated several times, for instance 1, 2, 3 or 4 times, especially once.

[0095] In the step e), the hydrogel is added at a temperature suitable to secure complete embedding of the initial cells in the hydrogel. After the addition, the plate is incubated to allow solidification of the hydrogel (e.g., at about 37°C for about 5-15 minutes). In a preferred aspect, the hydrogel is a hydrogel derived from natural extracellular matrix (ECM) such as Matrigel.

[0096] In the step f), appropriate media, and the combinations of nutrients and proteins, such as growth factors and morphogens, are added to the culture homogenously to guide the growth and development of the organoids. The media and the necessary nutrients, growth factors and morphogens are well-known by the person skilled in the art (e.g., Hans Clevers. Cell, 127:469-80 (2006) doi: 10.1016 / j.cell.2006.10.018; Toshiro Sato, et al. Nature. 459: 262-5 (2009); Greggio, C., et al, JoVE 51725 (2014) doi:10.3791 / 51725; Ivan Bedzhov and Magdalena Zernicka-Goetz. Cell, 156:1032-44 (2014)).

[0097] During step f), the initial cells are cultured in situ to allow their aggregation into multicellular aggregates and the multicellular aggregates are placed in conditions suitable for organoid development.

[0098] In addition, the method further comprises a step of observing the organoids, for instance by microscopy. More specifically, the organoids are observed in situ. There is no step of removing organoids from microwells and of recovery of organoids. Alternatively, the organoids can be removed from microwells and recovered.

[0099] The present invention relates to an array of organoids obtainable or obtained by the method for the preparation of organoids as disclosed herein. Indeed, due to the particular design of the microwells, the organoids are anchored to the microwells and protrude from the microwells. According to our knowledge, it is the first time that this kind of organoids is described.

[0100] The present invention relates to the use of a device as disclosed herein for preparing an array of organoids; the use of an array of organoids for screening assay, in particular assay for quantitatively and / or qualitatively assessing organoid development or perturbations thereof, for assessing toxicity of compounds and molecules, for personalized medicine, or for screening medicament candidates.

[0101] In a particular aspect, the present invention relates to a method for quantitatively and / or qualitatively assessing organoid development or perturbations thereof by compounds and molecules, comprising a) seeding the initial cells into the microwells; b) promoting organoid development; c) monitoring an effect of compounds and molecules on the organoid development by comparing the organoid development in absence of compounds and molecules, wherein compounds and molecules are contacted with the initial cell before, during or after seeding of the initial cells or during or after step b).

[0102] The effect can be monitored by measuring the size, the shape, the cellular composition and / or the phenotypic changes of the organoids. For instance, a specific response with biomarkers can be measured with quantitative fluorescent microscopy, e.g., by averaging the mean signal per condition and per compound, per cell within the organoid.

[0103] In the context of personalized medicine, the initial cells or some of them are provided from a tissue biopsy sample from a patient.

[0104] Definitions

[0105] By the term "about" is intended to mean the value more or less 10 % thereof, preferably 5 % thereof. For instance, about 10 means between 9 and 11, preferably between 9.5 and 10.5.

[0106] A microwell is a cavity capable of holding liquid, comprising an opening, a hollow space and a bottom. A microwell can also be referred to as a well or cavity. Bottom of microwells can be flat or round (e.g., U-bottomed). The cavity of a microwell can be cylindrical or prismatic.

[0107] The microwells are defined by a depth, a length and a width. The microwells are also defined by surface area.

[0108] By "top" of microwells is intended the upper part of the microwells comprising the opening of the microwells. By "bottom" of microwells is intended the lower part of the of microwells, opposite to the top.

[0109] The depth, also called the height, is the distance between the top and the bottom of the microwell.

[0110] The shape of the microwell is defined by viewing the microwell from the top of the plate. It can be defined by a length and a width, where the length is the longest dimension of the shape and the width is the smallest dimension of the shape. When the microwell is a round or is substantially round, the microwell can be defined by a diameter.

[0111] Surface area of a microwell is the area of the surface covered by the microwell by viewing the microwell from the top of the plate.

[0112] By "initial cell", it is referred to the cells that are seeded into the microwells. Stem cells are understood herein as cells capable of forming an organoid.

[0113] An array as used herein is defined as an ordered arrangement of similar or identical objects. Typically, the objects in an array can be divided into rows and columns. An array of organoids is an ordered arrangement of at least one organoid. In biology, arrays of samples or biological materials (microarrays) are used for high-throughput analysis.

[0114] A focal plane is the plane or flat surface through the focus perpendicular to the axis of a lens of, for example, of a microscope, especially fluorescent microscopy. At a particular focus, all objects in view are within the same focal plane.

[0115] High-throughput screens and assays are those which are automated to achieve levels of repeatable data acquisition unfeasible using manual methods.

[0116] A hydrogel (gel) is a 3D matrix comprising a network of hydrophilic polymer chains.

[0117] Matrigel is a commercial product widely used in both 2D and 3D models of cell culture. It comprises a solubilized basement membrane preparation extracted from an extracellular matrix rich mouse tumor.

[0118] Cultrex BME is a laminin-rich extracellular matrix that is secreted by the Engelbreth-Holm-Swarm tumor line (Li et al, 1987).

[0119] Myogels are extracellular matrices extracted from skeletal muscle. Cartigel is an extracellular matrix extract of cartilage.

[0120] In the present application, the term "organoid" designates three-dimensional culture systems of organ-specific cell types that develop from stem cells or differentiated cells and self-organize (or self-pattern) through cell sorting and spatially restricted lineage commitment in a manner similar to the situation in vivo. As used herein, an organoid is defined as a 3D culture of stem cells and their differentiated progeny or differentiated cells and their progeny, initiated from a single cell or a multicellular aggregate of cells with at least one stem cell (that is, an embryoid body). Stem cells and differentiated cells may be isolated from tissue or organoid fragments. Organoids grown from isolated intestinal crypts or stem cells may also be referred to in the field as "enteroids" or "colonoids". Organoids grown from or containing cancerous cells are "tumoroids". Optionally, the organoid can be a tumoroid. "tumoroid" refers to a cell aggregate that can mimic 3D cellular arrangement in a tumor.

[0121] Organoids are distinct from embryoid bodies at least in that organoids are self-organizing and thereby become architecturally similar to an in vivo tissue / organ, whereas embryoid bodies are not. Indeed, in order to obtain organoids from embryoid bodies, the embryoid body must typically be treated with patterning factors to drive the formation of the desired organoid identity. Optionally, the organoid can have a cystic structure with differentiated cells. Optionally, the organoid may include a lumen (a lumen being a cavity filled with fluid). Alternatively, the organoid may be devoid of lumen.

[0122] In situ is a biological term for culturing cells or tissues without moving their position.

[0123] BRIEF DESCRIPTION OF THE FIGURES

[0124] Figure 1: Experimental set up for the control of initial cell number. Fig la. Design of cavities for 1 initial cell. Fig lb. Design of cavities for 4 initial cells. For a and b, top: design of motif and bottom: the corresponding experimental images, Scale bar 5 pm. Fig lc. Cell diameter distribution. Top: Middle plane of single cells for MDCK, mES, pancreatic progenitor from top to bottom labeled with Sir-actin. Bottom: diameter distribution: N=3, n(MDCK)=75, n(mES)=202, n(pancreatic progenitor) =35. Scale bar 5pm. Fig Id. Mask for the design of the cavity map. Cavities of different diameters were arrayed and designed to accommodate different initial cell numbers. Scale bar 500pm.

[0125] Figure 2: Controlled MDCK cyst growth. Fig 2a. Typical dynamics of MDCK cysts forming from controlled initial cell numbers (E-cadherin in green, Podocalyxin in red). Initial cell numbers are shown at time 0 in cavities and the shapes of the same spheres are captured every day. Scale bar 10pm (N=3, n>10). Fig 2b. Bright field imaging of MDCK cyst growth inside cavities on the same focal plane with different time points. Fig 2c. Comparison of MDCK cyst growth at day 5 between the conventional culture condition (top panel) and inside cavities (middle panel). Bottom panel: quantitative comparison of day 5 MDCK cyst area between conventional culture (n=96) and microcavities (n=88). Fig 2d. MDCK cyst shapes can be controlled with cavities shapes. For Fig 2b, c and d, Scale bar 50pm.

[0126] Figure 3: Optimization of cell seeding and organoids growth (coating and sample design). Fig 3a. Phase contrast images of initial condition after cells seeding for different laminin concentrations. Fig 3b. The comparison between fibronectin and laminin coating on phenotypes of MDCK cysts. Fig 3c. The design of Matrigel reservoir. Top: scheme of cavities with and without Matrigel reservoir. Bottom: images of cavities with and without Matrigel reservoir.

[0127] Figure 4: Effect of cavities height on initial cell seeding and MDCK cyst growth. Fig 4a. Full coverage of cells in cavities at seeding is difficult to achieve when the height is much smaller than cell height. Images: representative images initial condition with height cavities 5pm (left, n=157) and 15pm (right, n=42). Fig 4b. Quantitative comparison of initial cell seeding between height cavities 5pm and 15pm. Fig 4c. Initial cells number is difficult to count when the cavities height is much larger than cell height. Fig 4d. MDCK cyst reached smaller size for cavities with height 80pm than 15pm with similar initial cells number. Fig 4e. More cells death was observed when using cavities with height 80pm (caspase 3 label). Scale bar 50pm.

[0128] Figure 5: Drug treatment. Fig 5a. The camptothecin treatment on MDCK cyst inside standard Matrigel culture and inside cavities. The position of MDCK cyst inside cavities before and after treatment. Fig 5b. The effect of different camptothecin concentrations on MDCK cyst. Scale bar 20pm.

[0129] Figure 6: Control of mES epiblast growth. Fig 6a. Typical dynamics of epiblast forming from controlled initial cell numbers (E-cadherin in green, Podocalyxin in red). Initial cell numbers are shown at time 0 in cavities and shapes of the same spheres are captured every day. Scale bar 10pm. Fig 6b. Time-lapse of epiblast with bright field images. Top: control condition with epiblast growth inside Matrigel standard culture. Bottom: epiblast growth inside cavities on the same focal plane. Time in hh:mm. Fig 6c. Quantitative comparison of epiblast area between control condition (n=24) and growth inside of cavities (n=12). Fig 6d. Effect of large initial cells number on epiblast phenotypes. Left: phase contrast images of initial cell number. Right: the corresponding epiblast 4 days after seeding with F-actin readouts.

[0130] Figure 7: Optimized Cavity Filling and Morphological Consistency in mES Cell Differentiation . Fig. 7a. mES Viable Cells Seeded at Day 0 . Fig. 7b. mES Epiblasts at Day 3. Fig. 7c. Cavity Occupation by mES Cells: Day 0 Seeding vs Day 3 Growth. Fig. 7d. Robust Circularity Consistency in mES Epiblasts (Day 3)

[0131] Figure 8: Control of other types of organoid growth. Fig 8a. Typical evolution of pancreatic spheres shapes as a function of initial cell number and time (F-actin in green, nucleus in magenta) (N=3, n>20). Scale bars 10pm. Fig 8b. Snapshots of intestine organoids at different time points. Fig 8c. Snapshot of Huh7 spheroids from human hepatocyte-derived carcinoma cells. Top: control condition growth inside Matrigel standard conditions. Bottom: Huh7 spheroids growth inside cavities and tracking of the same spheroids over time. Scale bar 20pm.

[0132] Figure 9: Comparison of MDCK cyst growth between two different types of wells. Fig 9a. Top: scheme of hydrogel well design. Bottom: the corresponding phase contrast images of MDCK cyst at different time points. Fig 9b. Top: The design of our cavities. Bottom: the corresponding phase contrast images of MDCK cyst at different time points. Fig 9c. The quantification of initial cells number for seeding inside hydrogel well (n=501) and cavities (n=181). Fig 9d. The quantitative comparison area of day 7 MDCK cyst between hydrogel well (n=147) and cavities (n=116). EXAMPLES

[0133] Organoids are 3D cellular systems in vitro which reproduce in vivo organs. They are relevant substitutes for standard 2D cell culture with potential impact on basic research and on pharmaceutical and medical applications. However, their preparations are poorly controlled and reproducible. Here, the inventors propose a device and a method which allow to control initial cell numbers at plating using microfabricated cavities. They showed that a fixed number of cells can be trapped at plating and this leads to the formation of organoids with reproducible shapes. Also, the device of the present invention allows to trap all cysts in the same observation plane which facilitates large scale content screening. The inventors reported the setup, its application for several cellular systems and quantified its performance compared to other systems. They also detailed the key steps in the protocol which are needed to obtain the target reproducible shapes, such as the height of cavity and its coating. Altogether the device and method of the invention offer rationalized approach to control organoid growth.

[0134] Cells have been studied for a century on flat Petri dishes. Many types of cells were plated at low density and they proliferated until layers reached confluency. This approach has allowed to reveal a large variety of basic phenomena (Kapalczynska et al., 2016; Liu et al., 2012) as well as new strategies to cure (Amelian et al., 2017). However, this method leads to artifacts (Belfiore et al., 2021). Cells grow in 3D in physiological conditions and they are rarely as spread as they appear on Petri dishes. In this context, 3D cell culture has emerged as a new method to replace the methods of culture.

[0135] Three dimensional aggregates, cysts and organoids offer promising tools to study any organs (Lancaster and Knoblich, 2014; Sato et al., 2009; Takasato et al., 2015) or diseases (Lancaster and Huch, 2019) such as cancer (Calandrini et al., 2020; LeSavage et al., 2022). Cells are embedded in a Matrix and they proliferate and self-organise over time into 3D systems which recapitulate the morphology and function of the corresponding organs. A large variety of organoids were prepared: pancreas (Greggio et al., 2014 and 2013), intestine (Sato et al., 2009), tumoroids (Sachs et al., 2018) etc... However, they are often poorly reproducible in shapes (Doth et al., 2022). Cysts overlap and they have various dimensions. Also, they are distributed over many planes of observations which limit automatised acquisitions for screening.

[0136] The inventors reasoned that if they controlled the initial cell numbers at plating, this could lead to reproducible shapes and size. So, they designed 3D microfabricated cavities allowing to trap a fixed number of cells (Fig. 1 and Materials and Methods). They could trap single cells, 4 cells or n cells. This control was made possible by the measurements of cell diameter when spherical in suspension (Fig. lc). Several systems were quantified (MDCK, mES cells, and pancreatic cells). Also, in order to track the same cysts over time, the inventors designed an array of cavities which allowed to return to the same points over time (Fig. Id). With this strategy, they could then follow over time in cell systems growing in 3D by using the associated protocols (see Materials and Methods).

[0137] The inventors reported that growth of MDCK cysts were dependent on the initial cell number (Fig. 2a). Even though the number of generated lumens was dependent on initial conditions, the final state was a single lumen. In addition, the generated cysts were remarkably reproducible in space and time (Fig. 2b). This was in sharp contrast with standard 3D culture in Matrigel (Fig. 2c). The inventors compared the distributions of cysts dimensions between both methods and they showed that their method provides a peaked distribution of sizes (Fig. 2c). Interestingly, they could also control the shapes of cysts by adjusting the cavities shapes, such as disks, triangles, ellipses (Fig. 2d). These experiments illustrate that microfabricated cavities allow to control initial cell numbers as well as control growth of cysts over time with reproducible shapes and organised array in the same observation plane.

[0138] This result was possible thanks to several steps in the protocol (Material and Methods). First, the inventors needed to optimise the extracellular matrix concentration to prevent spreading of cells away from cavities (Fig. 3a). Second, they also needed to select the right extracellular matrix protein to prevent spreading of cells outside cavities (Fig. 3b). Third, they had to optimise even distribution of Matrigel in 3D to allow homogeneous growth of cysts throughout the sample (Fig. 3c).

[0139] The height of cavities was also essential. The inventors showed that if cavities were too small, the trapping was poor (Fig. 4a, b). In addition, if height of cavity was too large, cell numbers could not be assessed (Fig. 4c). This large height was also deleterious for the cyst growths, since cells could undergo apoptosis in this condition (Fig. 4d,e). Altogether, the present results show that the cavity height has to be about the cell height to control initial cell numbers and their growths.

[0140] To further compare the approach of the inventors with standard methods, they grew cysts in controlled conditions and in cavities (Fig. 5). They used camptothecin which induces apoptosis. In controlled conditions, cysts overlapped and had distinct sizes in contract to our methods. Also, the drug treatment triggered simple changes in shape compared to standard growth conditions (Fig. 5a). The staining for caspase 3, a marker for apoptosis, confirmed this result (Fig. 5b). These experiments demonstrate that cavities allow to generate reproducible cysts with simple response in the presence of drugs, which open new strategies for drug screening.

[0141] To explore the relevance of the inventors' approach, they tested the same experiments with mouse embryonic stem cells (Fig. 6). They could control initial cell number (Fig. 6a) after measurements of cell diameter. The generated epiblasts were peaked in sizes compared to standard growth conditions (Fig. 6b, c). Finally, they could also control initial cell numbers with a large number such as 100, and epiblasts were generated with inner structures self-organised.

[0142] Building on these initial findings, additional data were generated to further demonstrate the robustness of the technique across a larger sample size. This extended analysis highlights the method's consistency and reliability in supporting mES cell seeding and growth. Representative images on large scale screening show viable mES cells at Day 0 and epiblasts at Day 3 (Figure 7a, b). The proportion of cavities occupied by viable cells at seeding (Day 0) and after growth (Day 3) reached respectively 100% and 88%, demonstrating effective seeding and robust growth conditions (Figure 7c). Morphological consistency of epiblasts was confirmed by a low coefficient of variation in circularity (5%), indicating homogeneous morphology (Figure 7d). These results suggest the method's potential to support consistent outcomes in cell seeding, growth, and morphological uniformity. These results show that the inventors' method works also for obtaining uniformed and controlled mouse epiblast in vitro from embryonic stem cells.

[0143] The inventors further tested the method on other cells. They cultured pancreatic progenitors isolated from E13.5 mice embryos (Material and methods). They obtained the same control and subsequent growth (Fig. 8a). The same results were obtained for intestinal organoids (Fig. 8b). Finally, they obtained homogenous and controlled hepatocytes spheroids with the methods of the invention in a reproducible manner (compare Fig. 8c top and bottom).

[0144] Alternative wells with large depth could be envisioned to culture organoids (Brandenberg et al., 2020; Hoehnel et al., 2019). To compare the performance of the inventors' methods with this strategy, they plated the same cells in each device (Fig. 9a, b). The inventors obtained a better control of initial cell numbers (Fig. 9c), as well as a much better control of cysts dimensions (Fig. 9d). This shows that the microfabricated cavities designed by the inventors have the ability to trap a fixed number of cells efficiently to secure growth of cysts and organoids with peaked dimensions.

[0145] The inventors have shown that trapping fixed initial cell number with microfabricated cavities allows to determine initial conditions. In turn, this generates 3D structures with regular shapes in the same plane. This approach could be generalised to any cellular systems because the design of cavities is based on cell dimensions.

[0146] Materials and Methods

[0147] Cell sources and expansion

[0148] The inventors used 5 different types of organoids or spheres starting from distinct cells, i.e., MDCK cyst, mouse epiblast, pancreatic sphere, Huh7 spheroids and intestinal organoids. They require specific medium for their growth. The MDCK II cell lines were cultured in MEM (Gibco 41090-028) with 5 % Fetal Bovine Serum (Sigma, USA), ImM Sodium Pyruvate (Gibco 11360-070) and lx NEAA (Gibco 11140050). MDCK II cells were resuspended every 3 to 4 days with trypsin-EDTA after they reached 70-95% confluency. A seeding density of about 5*105cells per 75 cm2was used for sub-culture. R1 ES-cell line was used for the culture of epiblast ((Martin-Lemaitre et al., 2020). Mouse embryonic stem cells were expanded with 1:1 DMEM / F12 (ThermoFischer 31331028) and neurobasal medium (Gibco 21103049) supplemented with lx N2 (Gibco 17502048), lx B27 (Gibco 12587010) and lx NEAA (Gibco 11140050), 55pM 2-Mercaptoethanol (Gibco 21985023), 3pM CHIR 99021 (Sigma SML 1046-5mg) and 2x LIF produced at IGBMC-Strasbourg in non-adhesive flasks (suspension culture flask, Cellstar 658195) (Martin-Lemaitre et al, 2020). Cells were sub-cultured every 4 days until the size of spheres reached a diameter of 80pm. Pancreatic spheres were prepared from the dissection of E13.5 embryos (mouse CD1 from Charles River Laboratory) using the protocol reported in Greggio et al (2014) and used without passaging. Human hepatocyte-derived carcinoma cells (Huh7) were cultured in DMEM (4,5g / l glucose, Gibco 11965092) with 10% Fetal Calf Serum (Sigma, USA), lx AANE, 1 mM Sodium Pyruvate and 40 pg / mL Gentamycin.

[0149] Organoids and cyst growth in Matrigel

[0150] Cells were diluted, around 105cells into 200 pl medium and then the cell suspension was diluted in chilled Matrigel (Corning, 356231) at a 1:3 ratio. Then we transferred 15pl mixing containing Matrigel, medium and cells. After polymerization of Matrigel in the incubator for 10 minutes, the relevant media was added according to cells or organoid types.

[0151] Cells diameter measurement

[0152] For the three systems, single cells were plated after trypsinisation and labeled using lOnM SiR-actin (TEBU-BIO, 251SC001). The middle planes of spherical cells were imaged. The associated surfaces were measured with Fiji and the distributions of cells diameters were plotted. Cavities diameters were designed accordingly by taking the mean value of each distribution to control the initial cell numbers (Fig. lc). All cavities had a cell height from 10pm to 17pm to keep the height similar to one cell diameter.

[0153] Microfabrication and cavity map

[0154] The inventors designed the samples with a map of patterns for cavities in order to: (i) track the evolution of the same cysts up to a week, (ii) test a large number of cysts with the same initial cell number and (iii) test the effects of different initial cell numbers for the same biological repeat (see Fig. la). The same strategy was adapted for each system by designing the cavity map accordingly.

[0155] Cavities were prepared using soft lithography as described in Bhat et al (2020). Briefly, they designed a mask with AutoCad to obtain a large number of motifs and different diameters. The motifs were selected to contain many initial cells number conditions. They used the following calculation of the motifs diameter, rescaled with the mean cell diameter: S(surface of cavities)= number of initial cells * surface of cells. These designs were printed on photomask and then these patterns were transferred on SU-8 silicon wafer with soft lithography. Next the design was replicated on a PDMS mold. Finally, these designs were transferred to cover-glass which allows us to achieve higher resolution images.

[0156] Cell seeding in microfabricated cavities for the control of initial cell number

[0157] To generate cysts and organoids in micro-fabricated cavities, the inventors seeded cells in microcavities with the following steps based on a former protocol (Bhat et al, 2020; Wollrab et al, 2016). Briefly, (i) the microfabricated-cavities on coverslips were activated with O2 plasma (Diener); (ii) substrates were incubated with 5pg / ml laminin (Sigma 11243217001) for 1 hour at room temperature followed by washing steps; (iii) cells in suspension were centrifuged 3 times at 1000 rpm for 3 minutes on the samples to direct cells inside micro-cavities; (iv) the coverslips were then rinsed gently to get rid of the excess of cells between cavities; (v) 15 pl Matrigel (Corning, 356231) was added on top of the sample. After solidification of the Matrigel, the relevant media were added depending on the cyst types. For pancreas spheres, single cells were dissociated from the E13.5 pancreases and immediately seeded in the micro-well without centrifugation.

[0158] System-specific media were added to obtain pancreatic spheres or epiblasts. Pancreatic sphere was formed by using DMEM / F12 (ThermoFischer 31331028) with B27 (Gibco 17504-044), recombinant Human FGF2 (R&D 233-FB-025), Y-27632 (Sigma Aldrich abl20129) and Penicillin-Streptomycin (Gibco 15070-063). Epiblasts were differentiated by using DMEM / F12 (ThermoFischer 31331028) and neurobasal medium (Gibco 21103049) containing 0.5x N2 (Gibco 17502048), lx B27 with vitamin A (Gibco 12587010), lx NEAA (Gibcol7504044), O.lmM 2-Mercaptoethanol (Gibco 21985023), 0.15mM Sodium Pyruvate and 0.2mM L-Glutamine (Life Technology GmbH 11360039). Mouse intestinal organoids culture method was detailed by Stemcell Technologies (Stemcell Technologies 70931).

[0159] Immunostaining

[0160] For immunostaining, the inventors followed standard protocols Greggio, C. et al (Greggio et al, 2014 and 2013). Briefly, samples were washed with PBS and fixed with 4% paraformaldehyde (PFA) diluted in PBS for 15 minutes. Cells were permeabilized with 0.5% Triton-X-100 for 15 minutes and then a blocking solution made of 1% NGS in PBS IX was added overnight. Primary antibodies were added directly to the blocking solution for two days at 4°C. Following 3 successive washing steps with PBS, the samples were stained with the relevant secondary antibodies for 2 hours at room temperature. The inventors used the following primary antibodies: Anti-E-cadherin (Abeam, Abll512), Phospho-Myosin Light Chain 2 (Cell signaling technology, #3674), Anti-Paxillin (Abeam, Ab32084), Alexa FluorTM Phalloidin 488 (ThermoFisher, A12379) for F-actin and DAPI (Sigma MBD0015) for the nucleus. Samples were washed three times in PBS and mounted on a homemade sample holder system for imaging and conservation.

[0161] Microscopy

[0162] In order to track the number of lumens in MDCK cysts, MDCK cells expressing Ecad - Podxl were used to visualize adherens junctions and apical side, respectively. After cell seeding, images were taken at an interval of 24 hours using Leica DM18 with an Evolve 512 camera coupled to a spinning disk microscope (CSU Wl) with a 25x water objective (NA = 0.9) and 63x glycerol objective (NA = 1.2) using the software Metamorph for image acquisition. Positions were chosen based on initial cell numbers and the same MDCKII cysts were acquired in 3D every 24h. This was possible with the cavity map reported above (Figure Id). For pancreatic spheres and epiblasts, initial conditions were controlled with the same method and samples were fixed and stained with the relevant antibodies before 3D acquisition with the same microscopy setup.

[0163] Imaging and data analysis

[0164] To extract area of organoids, the inventors used Fiji. Analysis and quantification were performed with GraphPad Prism 8.

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Claims

CLAIMS1- A device for preparing an array of organoids comprising a plate and a microfabricated substrate comprising an array of microwells designed for controlling number of initial cells seeded into the microwells, wherein the microwells are defined by a depth, a length and a width; the microwells have a depth of about one cell diameter of the initial cells; surface area of the microwells depends on the number of initial seeded cells; ratio of length and width of the microwells is less than 2, preferably about 1; the width or length of the microwells is greater than the depth; the distance between microwells is large enough that the organoids are not in contact with each other, the microwells are coated with a composition comprising adhesion protein(s) or peptide(s); and optionally, a subset of microwells is surrounded by walls to cover the subset of microwells with a hydrogel, especially a hydrogel derived from natural extracellular matrix (ECM).

2. The device of claim 1, wherein the surface area of the microwells is equal or substantially equal to the number of initial seeded cells * surface of one initial cell.3- The device of claim 1 or 2, wherein the microwells are coated with adhesion protein(s) or peptide(s) comprising at least 50%, 60%, 70%, 80% or 90% of laminin.4- The device of any one of claims 1-3, wherein the microwells depth is constant and within the range of 8 to 35 pm or of 10 to 30 pm or of 10 to 25 pm or of 10 to 20 pm or of 10 to 17 pm.5- The device of any one of claims 1-4, wherein the surface area of the microwells is within the range of 150 to 96,000 pm2, preferably from 150 to 10,000 pm2; and / or the shape of the microwells are selected from round, square, triangle, oval, diamond, rectangle, hexagon, and octagon.6- The device of any one of claims 1-5, wherein the device comprises an array of identical microwells or multiple arrays of identical microwells, each array having a microwell surface area adapted for seeding a different number of initial cells, and optionally at least 2, 3, 4, 8, 16, 32 cells or more, optionally less than 300 initial cells.7- The device of any one of claims 1-6, wherein the device comprises microwells further comprising the initial seeded cells in the microwells and optionally covered by the hydrogel, especially a hydrogel derived from natural extracellular matrix (ECM).8- The device of any one of claims 1-7, wherein the device comprises microwells further comprising organoids, said organoids being anchored into the microwells and protruding from the microwells.9- The device of any one of claims 1-8, wherein the plate comprises an array of wells comprising one or several arrays of microwells as defined in claims 1-8.10- Use of a device according to any one of claims 1 to 7for preparing an array of organoids.11- A method for preparing an array of organoids, comprising: a) providing a device according to any one of claims 1 to 6; b) contacting the device with initial cells to be seeded, the upper side of microwells being up; c) centrifugating so that cells are placed into the microwells; optionally repeating step c) at least once or twice; d) washing to remove cells remaining outside the microwells; e) optionally adding a hydrogel, especially a hydrogel derived from natural extracellular matrix (ECM), to cover the seeded microwells so that the initial cells are embedded into the hydrogel; and f) placing the device in conditions suitable for growing the initial cells and forming organoids.12- The method of claim 11, wherein the method comprises between steps c) and d) a step of incubating the device so that cells adhere to the microwells.13- The method of any one of claims 11 to 12, wherein the method comprises a preliminary step of determining the diameter of the initial cells, choosing a number of initial cells by microwells or a combination of numbers of initial cells by microwells and selecting a device having the appropriate size of microwells.14- The method of any one of claims 11 to 13, wherein the method further comprises a step of observing the organoids, for instance by microscopy.15- An array of organoids obtainable or obtained by the method of any one of claims 11 to 13.16- A kit for preparing an array of organoids comprising a device according to any one of claims 1 to 9.

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