Micro-droplet plate

Abstract

The object of the present invention is to provide a microdroplet plate (1) for the formation, separation, seeding and controlled recovery of a fluid of a volume of 1 to 400 microliters or 4 to 50 microliters, wherein the fluid is SOL, comprising: at least one stationary element (2) which is a rigid, flat support having an upper surface (14) and a lower surface (15), the stationary element (2) having on the upper surface (14) a plurality of wells (4) connected to each other by microchannels (5), the wells (4) being defined by a hydrophobic rim (21) and intersecting with holes (6), the wells (4) and microchannels (5) defining at least one microfluidic circuit (16); and a transfer element (3) which is a cylinder having an upper base (7) and a lower base (8) and is received in the wells (4) through the holes (6). Here, the transfer elements (3) move along and optionally around their vertical axes (28), even independently of one another, and the upper base (7) of the transfer elements (3) comprises a hydrophilic surface with relief items (12).A further object is to provide a method for the formation, separation, seeding and / or controlled recovery of gel droplets (17), optionally containing biological material.

Description

Background Art

[0001] The accurate transport and storage of numerous independent droplets containing bioreagents and cell materials is one of the important steps for conducting reliable large-scale biological assays.

[0002] There is a need for biological tests that can be performed more quickly and easily in order to make the process more economical while maintaining its accuracy and reproducibility. This is due to the rapid increase in the number of diagnostic and research molecular probes, and the fact that multiplexing assays on a wide range of devices is informationally advantageous. Tests using cell samples that reproduce the microenvironment (structure, biomechanics, and biochemistry) of the tissues from which these cells are derived are increasingly needed to study new aspects of drug resistance.

[0003] These biological tests require or are enhanced by the availability of methodologies for: (i) Handling cells / particles, analytes, and reagents in liquid and gel phases, (ii) Controlled seeding of fluorescent / bioluminescent molecules / cells / particles, (iii) Controlled immobilization of the above cells / particles for analysis purposes, (iv) Maintaining cell viability and cell function over a period sufficient for analysis purposes, (v) Controlling the exposure of one or more active ingredients to evaluate their effects on cell samples.

[0004] Important progress has been made in efficiently and reproducibly dispensing liquid volumes on the order of microliters, enabling an increase in the number of samples and minimizing the biological samples required, which is an important factor especially in the case of materials collected from patients.

[0005] Organoids mimic the physiological, pathological, and therapeutic responses in vitro by reproducing the originating tissue on a small scale and in 3D. Therefore, the methodology using organoids has enabled the reproduction of complex systems in the laboratory in a reproducible manner, aggregating many advantages of complex organs, which has led to a significant increase in interest in the international scientific community. The characteristics of these systems have been found to be useful not only for studying the physiology of biological systems that could not be easily and / or economically investigated in the laboratory until now, but also for various pathological conditions. A further important advantage of organoids is represented by the possibility of testing the efficacy of active ingredients on biological systems in a high-throughput and rapid manner, resulting in a significant reduction in cost and an increase in the number of active ingredients that can be analyzed. The possibility of developing personalized therapies using organoids offers an interesting and unique alternative in the current drug development process, which often utilizes animal models that are difficult to develop, expensive, and often raise ethical issues.

[0006] However, the 3D tissue formation approaches available today are difficult to integrate into large-scale systems without losing 3D tissue function.

[0007] Among the proposed solutions, in 2014, Frey et al. (Nature Communications 5, Article number: 4250) described a system based on the "hanging drop" technique for spheroid formation. However, the system of Frey et al. that functions in a biological system in a liquid phase is not applicable to cultures in solid and semi-solid media and cultures in independent droplets, such as those required for studies using organoids. In addition, in their "hanging drop" system, physical separation of the generated droplets is not achieved, eliminating the possibility of creating technical replicates of individual treatments, which is an essential requirement in large-scale studies.

[0008] U.S. Patent Application Publication No. 2017 / 0298314 describes a plate for depositing droplets onto a gel phase consisting of a series of elongated struts extending from a plane. The geometric shape of the elongated struts is advantageous for forming droplets at their upper ends and thus acts as an economical dispenser for the droplets themselves.

[0009] There is a strong demand for an economical and mechanically robust system for forming and dispensing gelatinous droplets on a large scale and uniformly, with volumes of 1 to 400 microliters or 4 to 50 microliters.

Summary of the Invention

[0010] The subject of the present invention is a micro-droplet plate, where a micro-droplet plate means a device suitable for the formation, separation, seeding, possible maintenance in culture, and controlled recovery of a fluid with a volume including 1 to 400 microliters or 3 to 50 microliters, preferably 4 to 30 microliters, and even more preferably 5 to 30 microliters, and the above fluid is Sol as follows.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

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Figure 8

Figure 9

Figure 10

Mode for Carrying Out the Invention

[0012] In the embodiment schematically shown in FIG. 1, the micro-droplet plate 1 according to the present invention comprises a fixed element 2 and a moving element 3 also called a piston. Referring to FIG. 1A, the fixed element is a rigid flat support having an upper surface 14 and a lower surface 15. On the upper surface 14, there is a series of wells 4 connected to each other by micro-channels 5. Each of the wells 4 is defined by a hydrophobic rim 21. The wells 4 intersect with holes 6. In one embodiment, the holes 6 occupy the entire base region of the wells 4. In an alternative embodiment, the holes 6 occupy a part of the base region of the wells 4. The base of the wells 4 is hydrophilic or hydrophobic, and in a preferred form, it is hydrophobic. The wells 4 and the micro-channels 5 constitute a microfluidic circuit 16. In one embodiment, the wells 4 have a flat bottom, and in an alternative embodiment, the wells 4 have a conical bottom, and the holes 6 occupy the apex of the cone.

[0013] Optionally, the fixed element 2 also comprises at least one access channel 13. The at least one access channel connects the lower surface 15 of the fixed element 2 to the microfluidic circuit 16. In one embodiment, the access channel 13 is inserted into one of the micro-channels 5, and in an alternative form, for example, it is inserted into one of the wells 4 by accessing through one of the side walls of one of the micro-channels 5 or one of the wells 4. In a preferred embodiment, referring to FIGS. 2A and 2B, the access channel 13 is inserted into an access well 23 located at a substantially central position of the support element.

[0014] In one embodiment, the holes 6 pass through the rigid flat support, and correspondingly, guide channels 24 appear from the lower surface 15 of the fixed element 2. In each of the holes 6, one of the moving elements 3 is received (FIG. 1B).

[0015] The moving element 3 is a cylinder made of plastic material with an upper base 7 and a lower base 8. The moving element 3 inserted into the hole 6 of the fixed element 2 moves along their vertical axis 28 and optionally rotates around their vertical axis 28. When inserted into the fixed element 2, the moving element 3 moves along its own vertical axis 28 and, independently of each other, takes variable positions between the rest position 9 and the working position 10.

[0016] In an embodiment comprising a guide channel 24, the moving element 3 inserted into the fixed element 2 through the hole 6 moves along its own vertical axis 28 which is conveniently received within the guide channel 24. The moving element 3 has an upper base 7 which, in the rest position 9, is in a position substantially in the same plane as the base of the well 4. That is, in the rest position 9, the upper base 7 of the moving element 3 closes the hole 6, making the well 4 similar to a closed well at the bottom.

[0017] The upper base 7 of the moving element 3 includes a hydrophilic surface having relief items 12. In one embodiment, when the moving element 3 is a cylinder with a circular base, the diameter of the upper base 7 is approximately equal to the diameter of the hole 6 which is likewise suitably circular. In this embodiment, the upper base 7 is the same hydrophilic surface having relief items 12. In a preferred embodiment, the moving element 3 is a cylinder in which the upper base 7 of area A rests on a base support 11 of area A', and the area A' of the base support 11 is larger than the area A of the upper base 7. In this embodiment, the moving element 3 is inserted through the hole 6 of the fixed element 2 and, in the rest position 9, the hole 6 is conveniently closed by the base support 11. In this embodiment, the base support 11 is a hydrophilic surface having raised elements 12.

[0018] Advantageously, the closing of each of the holes 6 operated by each of the moving elements 3 is a hermetic closing. Optionally, a suitable gasket is placed on the hole 6 to advantageously effect the above-mentioned sealing.

[0019] Thus, in the micro-droplet plate 1 with the moving element 3 at the stationary position 9, a series of wells 4 defined by the hydrophobic rim 21 are similar to the bottom-closed wells in the microfluidic connection therebetween, and the microfluidic connection is ensured by the microchannel 5 because its base consists of a hydrophilic surface having the relief item 12.

[0020] The micro-droplet plate with the moving element 3 at the stationary position includes an integrated microfluidic circuit 25 including the hydrophilic region of the micro-droplet plate. In one embodiment, the integrated microfluidic circuit 25 consists of the microfluidic circuit 16 and the hydrophilic surface having the relief item 12.

[0021] The relief items 22 on the hydrophilic surface having the relief item 12 are adjacent to each other and occupy 10% to 70% of the total surface, 40% to 60% in one embodiment, or 15% to 50%, preferably about 20%.

[0022] The relief items 22 are distributed on the hydrophilic surface in a controlled manner, that is, according to a regular geometric arrangement, and may be uniform with the same or different relief items arranged in the same pattern on the surface, or may be non-uniform with relief items of different shapes / sizes / distributions on the surface.

[0023] FIG. 3 shows, by way of example, some geometric shapes of the relief items 22. The figure schematically shows embodiments of the geometric shapes and distributions of the relief items. The relief items 22 shown in black in the figure are surrounded by cavities 26 shown in gray and interconnected in the figure. Advantageously, the relief items 22 do not have a vertical rim and have a rounded rim. In one embodiment, the relief items 22 are elongated elements (FIGS. 3A, D, E), circular elements (FIG. 3G), rod-shaped elements (FIG. 3B), arch-shaped elements (FIG. 3F), or a combination of elements having variable shapes among the elements shown (FIG. 3C). In a further embodiment, they are elongated curved elements extending radially (FIG. 3H).

[0024] The height of the relief item 22 is approximately equal to the depth of the well 4. In one embodiment, all the elements 22 on the hydrophilic surface having the relief item 12 have the same height. In one embodiment, the elements 22 on the hydrophilic surface having the relief item 12 have different heights independently of each other (FIGS. 9A, 9B, 9C). Preferably, the relief items 22 having a higher height are arranged around the hydrophilic surface (FIG. 9E). In this embodiment, the surrounding relief items 22 advantageously define its surface and Sol enable better control of the cap 27.

[0025] The surface on which the relief items 22 appear is optionally subjected to a surface treatment suitable for making it hydrophilic to the extent that it functions to fill the well.

[0026] The number of relief items 22 on the hydrophilic surface having the relief item 12 reaches an extent of the occupancy percentage of the surface that functions for the purpose, i.e., 10-70%, 40-60% or 15-50%, preferably about 20%.

[0027] In a preferred form, the relief items 22 are arch-shaped elements and elongated elements arranged on the surface along concentric circles as shown in FIG. 3C.

[0028] In one embodiment, referring to FIG. 8A, the geometric relief item 22 having an elongated or substantially circular shape has the minimum and maximum dimensions shown in Table 1. Here, D = diameter or equivalent diameter, SDa = characteristic distance in that direction between one element and another element, SDb = other characteristic distance in that direction between one element and another element (in the case of non-uniform distribution), LD = second characteristic dimension of the element (when not derived from a circular shape), H = height of the element.

[0029]

Table 1

[0030] In one embodiment, the rod-shaped or arch-shaped geometric relief item 22, referable to FIG. 8B, or the spiral according to FIG. 9D has the minimum and maximum dimensions shown in Table 2. Here, A is the measurement of the long side of the relief item, B is the measurement of the short side of the relief item, w is the distance from an adjacent element of one of these elements, H = height of the element.

[0031]

Table 2

[0032] The fluid loaded in the microfluidic circuit 16 fills the hydrophilic region in the microfluidic circuit by capillary action and forms droplets on the upper base of the moving element 3 or on the base support, where the size of the droplets is defined by the hydrophobic rim 21 defining the well and by the surface tension at the liquid / air interface.

[0033] The height of the relief item 22 is about 10 to 20% of the size of the droplet formed on the hydrophilic surface having the moving element 12. As an example, FIGS. 9A, 9B, 9C and 9E show Sol a droplet which is the cap 27, the droplet being formed corresponding to the base support 11 on the upper base 7 of the movable element 3, where the geometric relief items 22 on the hydrophilic surface having the relief item 12 have different heights from each other.

[0034] In one embodiment, the fixed element 2 and the moving element 3 are made of a thermoplastic polymer selected from the group consisting of polystyrene, polyolefin, polycarbonate and acrylonitrile-butadiene-styrene, or are made of polysiloxane, preferably PDMS (polydimethylsiloxane).

[0035] The fluid loaded into the micro-droplet plate according to the present invention is Sol a fluid that changes from a liquid phase to a solid gel by a phase transition.

[0036] The phase transition is caused by a physical stimulus (temperature, UV) or a chemical stimulus (for example, exposure to specific ions or solvents, change in pH).

[0037] In a preferred form, the Sol is a formulation that polymerizes to produce a biologically active matrix. As an example, it is a formulation based on alginate, chitosan, hyaluronic acid, fibrin, laminin, collagen or a combination thereof. Preferably, it is Matrigel (Corning® Matrigel®).

[0038] On the hydrophilic surface having the raised element 12 on the upper base 7 of each of the moving elements 3, when the moving element 3 is in the stationary position 9, a droplet of the fluid Sol ( Sol referred to as the cap 27 of) is obtained, which becomes the gel droplet 17 after an appropriate phase transition. In one embodiment, the SolIt contains organoids 18, which are uniformly distributed in the gel droplets 17.

[0039] When the gel droplets 17 are formed on a hydrophilic surface having each relief item 12 of the moving element 3, by moving the moving element 3 from the stationary position 9 to the working position 10, they are conveniently seeded onto a culture plate containing a growth medium. Once placed in the medium, the gel droplets remain, the structure of the organoids is preserved therein, and at the same time, the necessary exchange of metabolites between the organoids and the medium is ensured.

[0040] As an example, a molecule whose effect on the organoids is to be tested is added to the above-mentioned medium, enabling a large-scale drug screening test on the organoids.

[0041] Next, the micro-droplet plate according to the present invention will be described under the usage conditions. An overview of various working steps is shown in FIG. 4.

[0042] Step 1: Loading with micro-droplet formation (FIG. 4A) The micro-droplet plate according to the present invention is arranged upward with the moving element 3 in the stationary position 9 (upper panel in FIG. 4A), Sol and a fluid, which is, is loaded into the integrated microfluidic circuit 25.

[0043] The above-mentioned fluid is loaded from below or from above through the above-mentioned access channel 13. When loading through the access channel 13 arranged at a position substantially central with respect to the fixed element 2, the above-mentioned fluid is preferably introduced into one of the micro-channels 5 or into one of the wells 4 or 23.

[0044] In the case of upper loading, for example, a pipette, a syringe pump or an injector is used, and they are inserted into one of the wells 4 or the micro-channels 5.

[0045] Regarding Figure 10, in another embodiment, the loading is obtained by a dispenser 29 that is in fluid connection with the container 30. The dispenser is conveniently managed in an automated manner according to methods known to those skilled in the art and is arranged such that the released fluid reaches the selected wells (Figure 10A). Alternatively, the dispenser is arranged near the plate so as to directly release the fluid into the selected wells (Figure 10B). The automated movement of the dispenser conveniently enables subsequent dispensing in a plurality of wells.

[0046] Alternatively, the fluid is loaded from below through the access channel 13, for example using a pump system.

[0047] After loading, the fluid freely flows and occupies the hydrophilic regions present in the integrated microfluidic circuit 25, that is, the hydrophilic surfaces having the microchannels 5 and the relief items 12. Due to the hydrophilic surface having the relief item 12, the fluid expands above the wells and protrudes from the microdroplet plate. Sol Forms the cap of 27. When the integrated microfluidic circuit 25 is conveniently filled with the fluid, the fluid gels, and thus a microdroplet plate in which the hydrophilic regions of the integrated microfluidic circuit 25 are uniformly occupied by the gel is obtained. In particular, Sol The fluid seen corresponding to each of the hydrophilic surfaces having the relief item 12 that forms the cap of 27 gels to form gel droplets 17.

[0048] In a preferred embodiment, the microdroplet plate is oriented downward for the gelation process (lower panel in Figure 4A), and in downward gelation, it has been observed that gel droplets with a larger volume than those obtained by gelation that keeps the microdroplet plate upward are advantageously obtained.

[0049] In one embodiment, the base of the well 4 is hydrophobic and does not attract the fluid which is an aqueous fluid. Thus, the base of the hydrophobic well 4, and in addition any gaskets, further ensure the seal and the fluid remains on the hydrophilic surface having the relief item 12 defined by the hydrophobic rim 21.

[0050] The interconnected cavities 26 are advantageously such as to completely cover the hydrophilic surface with the relief item 12. In fact, the inventors of the present invention have surprisingly demonstrated that the geometry, distribution and number of the relief items 22 on the hydrophilic surface included in the upper base 7 of the moving element 3 enable the flow front to advance uniformly along each direction of the microfluidic circuit 16 until it completely occupies its hydrophilic area. A hydrophilic surface without the relief item 22, or a hydrophilic surface with too low a percentage (less than 10%) of the relief items, or a hydrophilic surface with too high a percentage (70%) of the relief items does not allow the above uniform filling. Under these conditions, it has actually been observed that the fluid does not reach some of the hydrophilic surfaces.

[0051] Step 2: Microdroplet insulation (Figure 4B) The microdroplet plate having the hydrophilic area of the integrated microfluidic circuit 25 uniformly occupied by the fluid in the gel phase state is downward and is conveniently arranged on the cell culture plate 20 containing the medium 19. The moving elements 3 loaded with the gel droplets 17, even if independent of each other, reach the working position 10 or appear from the fixed element 2 towards its upper surface 14.

[0052] Advantageously, according to the microdroplet plate according to the invention, the formation of the droplets is enabled by the hydrophobic rim 21 defining the hydrophilic area, so that the loading of the entire microdroplet plate can be carried out in a single operation. Thanks to the possibility that the moving element 3 moves along its own axis after the transfer to the gel, the gel droplets 17 appear from the plate and are accessible in the subsequent seeding step.

[0053] Advantageously, the cell culture plate 20 is a multi-well plate, and at least one well of the cell culture plate is conveniently found under the moving element 3 loaded with the gel droplet 17.

[0054] Step 3: Seeding (Figure 4C) The gel droplet 17 on the moving element 3 is immersed in the culture medium 19.

[0055] In one embodiment, the moving element 3 retreats from the working position 10 to the stationary position 9 to release the gel droplet 17 into the culture medium 19. Optionally, the moving element 3 is made to rotate about its own vertical axis 28, thereby facilitating the release of the gel droplet 17 into the culture medium 19.

[0056] The droplet 17 containing the cell material (e.g., organoid) is then held in the culture medium throughout the entire period of the treatment. In one embodiment, the micro-droplet plate and the moving element 3 are held in the seeding position throughout the entire period of the treatment. In this embodiment, the gel droplet 17 remains immersed in the culture medium and adhered to the hydrophilic surface having the relief item 12.

[0057] Advantageously, the moving element 3 of the micro-droplet plate according to the present invention enables rapid seeding of uniform gel droplets in the plate.

[0058] Step 4: Recovery (Optional) If the moving element 3 has been removed from the seeding position, it is returned to contact the surface of the culture medium 19 at the end of the treatment. The gel droplet 17 immersed in the culture medium re-adheres to the hydrophilic surface having the relief item 12.

[0059] If the moving element 3 has been held in the seeding position throughout the entire period of the treatment, the gel droplet 17 is already conveniently adhered to the surface having the relief item 12.

[0060] Optionally, the moving element 3 is removed, whereby the gel droplet 17 is collected from the culture medium 19. The collected gel droplet 17 is prepared for reuse, for example, by being exposed to different treatment protocols for subsequent seeding in different culture media.

[0061] Conveniently, the microdroplet plate according to the invention comprises M×N wells, where M is 2 or more and N is 2 or more. Preferably, M is 12 or less and N is 32 or less. In one embodiment, the multi-droplet plate comprises 3×3, 5×5, 7×7, 4×8 or 8×8 wells. Conveniently, the wells are distributed in the microdroplet plate while maintaining the same distribution shape as that found in a 96-well, 384-well or 1536-well multi-well plate.

[0062] Optionally, one or more microdroplet plates according to the invention are inserted into a support that accommodates the number of microdroplet plates necessary to seed an entire 96-well, 384-well or 1536 multi-well plate.

[0063] As an example, FIG. 5 shows a perspective view of a multi-well plate 20, specifically a 384-well plate, on which a series of microdroplet plates 1 according to the invention are arranged in a number sufficient to cover the entire wells.

[0064] In one embodiment, the moving element 3 is moved by an automated technique, and each of the moving elements 3 is controlled to move independently.

[0065] In one embodiment, the microdroplet plate comprises a block structure suitable for positioning the moving element 3 at a desired position, such as a stationary position 9 or a working position 10 or an intermediate position, independently of each other. Conveniently, the multi-well plate has an optically transparent bottom so that the internal volume of each well can be observed under a microscope.

[0066] The following examples are intended to better illustrate the solutions proposed in this specification and are not intended to be limiting in any way. The scope of the present invention is defined by the following claims.

Example

[0067] Example 1: Organoid Seeding in a 384-Well Plate The organoids were derived from tumor cells isolated from liver metastases collected during surgery from a patient with colorectal cancer. Cancer cells dissociated from the original tissue were resuspended in Corning Matrigel, a basement membrane preparation in a solution extracted from a mouse sarcoma rich in extracellular matrix proteins. The suspension was then seeded in the form of droplets and, after polymerization, grown in a medium. The composition of the medium consists of a defined combination of additives and growth factors, which has been studied and optimized for the efficient generation and long-term growth of three-dimensional organoids derived from human gastrointestinal cancer cells. Prior to drug treatment, the organoids were harvested, dissociated into single cells, and then resuspended in Matrigel at a concentration of 200 cells / μL. The Matrigel and cell preparation was then seeded onto conventional 384 multiwell or microdroplet plates and incubated for 20 minutes in a cell culture incubator at 37 °C and 5% CO2 to promote Matrigel polymerization, and then immersed in 24 μL of growth medium per well containing an appropriate amount of drug.

[0068] Figure 6A shows an exemplary photograph of a Matrigel droplet containing organoids formed in a microdroplet plate. Figure 6B shows an exemplary inverted microscope photograph of a microwell seeded with organoids using a microdroplet plate.

[0069] After seeding, the organoids were exposed to the following triple treatment. DMSO: Solvent only BTZ: 1 μM bortezomib OLA: 1 μM olaparib

[0070] After 96 hours of treatment, the viability of the organoids was measured with the CellTiter-Glo® 3D reagent (Promega) and reported as a percentage relative to the solvent-only treated control.

[0071] As shown in the graph of Figure 7, the cell viability of the organoids at seeding and after 96 hours (upper panel) is equivalent in wells seeded by the conventional method (A) and wells seeded by the multi-droplet plate (B). The same considerations apply to the measurement of the effect of the tested additives. The examples show that the solution according to the invention can give rise to an organoid culture equivalent to that obtained by the conventional method, without being on a large scale.

Explanation of reference numerals

[0072] 1 Micro-droplet plate 2 Fixed element 3 Moving element 4 Hydrophilic well 5 Micro-channel 6 Hole 7 Upper base 8 Lower base 9 Rest position 10 Working position 11 Base support 12 Hydrophilic surface with relief item 13 Access channel 14 Upper surface 15 Lower surface 16 Microfluidic circuit 17 Gel droplet 18 Organoid 19 Medium 20 Cell culture plate 21 Hydrophobic rim 22 Relief item 23 Access well 24 Guide channel 25 Integrated microfluidic circuit 26 Interconnected cavities 27 Sol cap of 28 Vertical axis 29 Dispenser 30 Container

Claims

1. A micro-droplet plate (1) for the formation, separation, seeding and controlled recovery of a fluid having a volume of 1 to 400 microliters or 4 to 50 microliters, wherein the fluid is a fluid (hereinafter referred to as "sol") that changes from a liquid phase to a solid gel by a phase transition, - at least one fixed element (2) which is a rigid flat support having an upper surface (14) and a lower surface (15), on the upper surface (14) having a plurality of wells (4) connected to each other by micro-channels (5), the wells (4) being defined by a hydrophobic rim (21) and intersecting with holes (6), the wells (4) and the micro-channels (5) defining at least one micro-fluidic circuit (16), the fixed element (2), and - a cylinder having an upper base (7) and a lower base (8), and including a moving element (3) received in the well (4) through the hole (6): a micro-droplet plate Here, The moving elements (3) can move along their vertical axes (28), even independently of each other, and optionally move around their vertical axes (28), The upper base (7) of the moving element (3) includes a hydrophilic surface having relief items (12).

2. The micro-droplet plate (1) according to claim 1, wherein the fixed element (2) further includes at least one access channel (13) connecting the lower surface (15) of the fixed element (2) to the at least one micro-fluidic circuit (16).

3. The moving elements (3) are in a stationary position (9) or a working position (10), even independently of each other. The moving elements (3) can reach the stationary position (9) and the working position (10) by moving along their vertical axes (28). When the moving element (3) is in the stationary position (9), the upper base (7) is in a position substantially on the same plane as the base of the well (4). When in the working position (10), the moving element (3) emerges from the fixed element (2) towards the upper surface (14) of the fixed element (2). The micro-droplet plate (1) according to claim 1 or 2

4. The micro-droplet plate according to any one of claims 1 to 3, wherein the moving element (3) is in the stationary position (9) and forms an integrated micro-fluidic circuit (25) including the micro-fluidic circuit (16) and a hydrophilic surface having relief items (12).

5. The hydrophilic surface having the relief items (12) includes relief items (22) that are distributed in a controlled manner across the entire hydrophilic surface such that they occupy 10 to 70% of the entire hydrophilic surface, the microdroplet plate according to any one of claims 1 to 4.

6. The microdroplet plate according to claim 5, wherein the relief items are uniformly distributed.

7. The microdroplet plate according to any one of claims 1 to 6, which is loaded with a sol that is a formulation that polymerizes to produce a biologically active matrix.

8. The microdroplet plate according to claim 7, wherein the sol is selected from the group consisting of formulations based on alginate, chitosan, hyaluronic acid, fibrin, laminin, collagen, Matrigel® (manufactured by Corning Inc.) or combinations thereof.

9. Use of the microdroplet plate according to any one of claims 1 to 8 for the formation, separation, seeding and / or maintenance and / or controlled recovery of gel droplets (17) optionally containing biological material in culture.

10. The use according to claim 9, wherein the gel droplets (17) are in Matrigel and the biological material consists of organoids.

11. A method comprising the following steps for the formation, separation, seeding and / or maintenance and / or controlled recovery of gel droplets (17) in culture: - Providing a microdroplet plate according to any one of claims 1 to 8, - Making a fluid that is a sol available, - Placing at least one moving element (3) in a rest position (9), - Loading the microdroplet plate with the fluid of the liquid phase that occupies the integrated microfluidic circuit (25) by capillary action, - Polymerizing the fluid to obtain gel droplets (17) at each of the hydrophilic surfaces having the relief items (12), - Seeding the gel droplets (17) by turning the microdroplet plate downward and moving at least one moving element (3) along its vertical axis (28) to reach a working position (10) to cause the gel droplets (17) to appear, - Optionally, rotating at least one moving element (3) around its vertical axis (28) to facilitate the release of the gel droplets (17). Optionally, relocate the hydrophilic surface having the relief item (12) into contact with the gel droplet (17) to return the gel droplet (17) to the microdroplet plate. **Claim 12** The method according to claim 11, wherein the gel droplet (17) contains a biological material, the downward-facing microdroplet plate is disposed above a cell culture plate (20) containing a culture medium (19), and the gel droplet (17) is seeded onto the cell culture plate (20). **Claim 13** At least one moving element (3) is held at the seeding position throughout the treatment, the gel droplet (17) remains within the culture medium (19) throughout the culture / treatment, adheres to the hydrophilic surface having the relief item (12), and optionally, at the end of the culture / treatment, the at least one moving element (3) is withdrawn along its vertical axis (28) to reach an intermediate position between the working position (10) and the rest position (9), and the gel droplet (17) containing the biological material collected from the culture medium (19) is available for reuse, according to the method of claim 12. **Claim 14** The method according to claim 12 or 13, wherein the biological material consists of an organoid (18).

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