Microstructured device and method of production thereof

Abstract

The present invention describes a microstructured device for the implementation, observation and characterization of organoids / spheroids as three-dimensional cell models suspended in time-lapse and / or confocal microscopy with immersion objectives, wherein these cell models are suitable for the study of organogenesis, carcinogenesis and drug screening. The invention is also related to the method of production of said device.

Description

[0001] MICROSTRUCTURED DEVICE AND METHOD OF PRODUCTION THEREOF

[0002] Description

[0003] Field of invention

[0004] The present invention refers to the field of technologies for the analysis and acquisition of images of live cells, suspended 3D cell models. In particular, the invention refers to a device designed to house and examine biological material ranging in size from millimeters to micrometers (also referred to below as a microstructured device). The device can also be used to obtain spheroids of tumor cells and to allow direct observation of three-dimensional cell models suspended in time-lapse and / or confocal microscopy with immersion objectives.

[0005] In the following, the terms device and microstructured device will be used interchangeably and are to be considered synonymous.

[0006] Known art

[0007] Organoids are miniature replicas of human organs and tissues, often derived from induced pluripotent stem cells (iPSCs) or from adult cells and tissues, comprising tumor-type cells. These are structurally less complex formations than an organ, but decidedly more representative than a cluster of cells arranged on a laboratory plate. In fact, unlike classical 2D culture methods, an organoid has the advantage of reproducing the three- dimensional and conformational structure of the original organ.

[0008] The ability of the cells that compose them to organize and distribute themselves in an orderly manner, following the most important steps of the organogenesis process, has made them unparalleled and very promising 3D cellular models for obtaining new information on the development of the various organs and the interactions between the tissues that form them.

[0009] These models have been widely used in biomedical research, to test new drugs (drug screening) or to study the processes of organogenesis or carcinogenesis.

[0010] Organoids can be formed from cells taken directly from patients and, therefore, are essential elements to understand what happens to an organ when it is attacked by a disease such as cancer. In addition, researchers are thinking that they can exploit them to assess the impact in terms of toxicity of a new drug on the physiology of various organs and, therefore, hope that organoids will soon become a useful link in the development chain of targeted therapies against cancer and many other chronic or autoimmune diseases.

[0011] In this context, several examples of devices are available that allow to obtain three-dimensional cultures, such as spheroids / organoids. Some of these comprise Microtissue 3D Petri Dish micromould spheroids (Merck KGaA© #Z764043, #Z764035, #Z764019, #Z764000, #Z764051 ), 96-well cell culture plates with low attachment surface from several companies, and microfluidic platforms or rotary flasks used for spheroid production.

[0012] However, there are currently no devices on the market that can be made in different sizes and with different structures according to the needs of use, while always maintaining the characteristic of being usable for observations with microscopes and objectives of various kinds such as inverted microscopes and / or microscopes with immersion objectives for the automated visualization of 3D cellular constructs, spheroids, scaffolds (cellularized structures) useful for preclinical research and for microscopic observation in time-lapse and / or confocal.

[0013] The currently existing platforms do not allow the immobilization of the sample (essential for these observation methods) and often, not being optically accessible, do not allow direct observation of the sample. Immobilization of the sample is impossible with today's devices because the mediums wherein the spheroids are housed are "floating" and move even due to minimal stresses. Instead, it is desirable to have a device wherein it is possible to insert a sample that is deposited, by gravity, in a cavity of the structure and remains there in a substantially fixed position even if hydrated.

[0014] In the case where fluorescence microscopy is used for the analysis of spheroids, organoids or tissues, i.e. an investigation technique wherein sample fixation, inclusion in resins and cutting, or disintegration or cytocentrifugation is used for the observation of individual cells, further limitations emerge. In fact, this technique involves the loss of many important information, such as the interaction between cells and the cell- extracellular matrix interaction, the relationship between living cells throughout the thickness of the sample, the three-dimensional localization of proteins, the spatial hierarchy of cells and the presence of possible vascular structures.

[0015] Commercially available devices for the observation or microscopy of three-dimensional samples have the following macroscopic problems:

[0016] • The need to move / handle the spheroids obtained on a special support, such as a slide for microscopic observation;

[0017] • The impossibility of conducting observations on live three- dimensional samples immersed in a culture medium through an immersion microscope. The existing devices are in fact made of hard plastic such as to put at risk a sensitive and precious equipment such as that of a microscope with an immersion objective. By using this microscope with immersion objective, in fact, the approach to a three-dimensional sample is avoided to avoid the impact and / or rubbing of the objective against the hard walls of the device.

[0018] Furthermore, with current commercial devices it is substantially impossible to obtain spheroids on a support and the consequent and / or simultaneous observation of the spheroids obtained and / or being formed on the same support.

[0019] Further common limitations that characterize the devices on the market for the microscopic observation of are the following:

[0020] • they cannot be made by the buyer but must be purchased ready- to-use from the supplier, they are not very user-friendly, and they are not reusable, with a consequent significant impact on costs and time of carrying out the exams; they are not very adaptable to the experimental needs deriving from the various types of experimental samples (e.g. spheroids, bioprints, cellularized scaffolds, PDT (patient-derived tissue) or tissue fragments taken from patients or animals) and are therefore only usable for a specific type of three-dimensional sample; they do not adapt to the different types of sample processing, such as the fixation and observation of samples that are still viable, and are often incompatible with the image acquisition systems commonly found in research / analysis laboratories, requiring the acquisition of specialized tools for analysis that may not be economically accessible to all users; they require the use of large quantities of reagents for fluorescence cell assays, resulting in a significant cost impact; they do not allow direct observation of live cells, with high quality of vision and resolution, since these systems are either closed or provide for the housing of the spheroids in rigid and narrow supports, preventing the use of immersion objectives; they do not always allow the immobilization of the sample, which is instead essential to make observations of cellular behaviour at regular time intervals or to make observations under the confocal microscope; they do not always offer the possibility of modulating the extracellular microenvironment within the system, both before and during microscopic observation, in order to better simulate a patient's organic tissue and improve the predictive capacity of the model used; they often do not allow the cultivation and observation of three- dimensional samples above 200-300 micrometers (200-300 pm, 10’6m). This represents an important limitation since the possibility of making / observing samples at the mesoscopic level, i.e. samples with dimensions preferably between 0.1 mm and 5mm, allows to create a cellular model closer to reality and consequently increases the predictive capacity of drug responses, allowing the formation of gradients, necrotic areas and hypoxic or acidic zones within the sample itself.

[0021] The 96-well cell culture plates with low attachment surface, which are the standard for obtaining spheroids and organoids, have limitations for confocal microscopy and time-lapse visualization. The limited depth of the wells (usually 10-11 mm) can prevent a complete acquisition along a depth Z axis, i.e. an axis perpendicular to the plane on which the plate is placed, of large spheroids, limiting confocal imaging in depth. The curvature of the bottom of the well can introduce aberrations and distortions into the image, compromising the quality of confocal imaging. The limited space between the top of the well and the confocal microscope objective can make it difficult to use the high-magnification, high-numerical aperture objectives required for confocal imaging. When time lapse acquisitions are made, i.e. automated acquisitions at multiple time points (wherein, by way of example, an image is acquired every hour, at regular intervals of minutes, every minute, at regular intervals of seconds, every second and / or at another interval, preferably for a total observation period of 24 hours), wherein it is required that the objects to be observed are always in the same position (x, y, z), further limitations are supplemented. Moving spheroids in wells can move out of the plane of focus during image acquisition, affecting imaging quality. In addition, quantitative analysis of parameters such as size, shape, presence of specific proteins, and proliferation can be impaired by spheroid mobility.

[0022] Unless specifically excluded in the detailed description below, this chapter is to be considered as an integral part of the detailed description of the invention.

[0023] Summary of the invention

[0024] The aim of the present invention is the implementation of a device as claimed in claim 1 and the claims dependent on it. Further aims and benefits will be evident from the detailed description of the invention below, and the claims describe preferred embodiments of the invention, forming an integral part of the present description.

[0025] The advantages offered by this invention are evident in the light of the description presented so far and will be even clearer thanks to the attached figures and the related detailed description.

[0026] Brief description of the figures

[0027] The invention will be described below in at least one preferred embodiment by way of example and not limitation with the help of the attached figures, wherein the following are shown:

[0028] Figure 1 . Top view of a device 100 according to the present invention;

[0029] Figure 2. Top view of a tray 210 according to the present invention;

[0030] Figure 3. Top view of a cap 220 according to the present invention;

[0031] Figure 4. Section of a cap 220 (FIG. 4A) and a device 100 (FIG. 4B) obtained with the same, according to the present invention;

[0032] Figure 5. Section of a cap 220 (FIG. 5A) and a device 100 (FIG. 5B) obtained with the same, according to the present invention;

[0033] Figure 6. Section of a cap 220 (FIG. 6A) and of the device 100 (FIG. 6B) obtained with the same, according to the present invention;

[0034] Figure 7. Some steps of a method of manufacturing a device 100 using a mould 200 according to the present invention;

[0035] Figure 8. Magnified views of surface cavities 101 of the device 100. These views are images obtained with a stereo microscope showing the cone- shaped cavity 101 of an agarose device 100 (top, 1x magnification with a black bar of the scale corresponding to a length of 2 mm; below, 2x magnification, black bar of the scale corresponding to a length of 1 mm). In the image above can be seen the detail of two rows of cavities 101 , in the image below can be seen the detail of a row of cavities 101 ;

[0036] Figure 9. Top views of a tray 210 and an imaging slide 2. Tray 210 shown at the top of the Figure is made of polydimethylsiloxane with the agarose device 100 inside. In the lower part of the Figure is shown said imaging slide 2 of standard size. It can be seen that the dimensions in plan (plane X, Y identified by the axes inserted as a reference in the Figure) of the tray are approximately equal to those of a standard imaging slide 2 (25x70 mm);

[0037] Figure 10. View of a PDMS tray 210 containing an agarose device 100. Said device 100 comprises three compartments 102 comprising cone- shaped cavities 101. A culture medium (200 pL) was inserted only in the first and third compartments 102 (compartments 102 closest to the external perimeter of the device 100) in order to show that the culture medium remains confined to compartments 102 allowing the creation of different cultures for each compartment 102;

[0038] Figure 11. Image of a polydimethylsiloxane tray 210, with agarose device 100 inside, placed in a slide slot of a confocal microscope. The plan dimensions (x, y plane) of the polydimethylsiloxane tray 210 are equivalent to the dimensions of the support for the observation of slides under the confocal microscope;

[0039] Figure 12. View of a polydimethylsiloxane tray 210, with an agarose device 100 in it, placed in a slide slot of an automated imaging fluorescence microscope (ImageXpress Pico). The plan dimensions (x, y plane) of the polydimethylsiloxane tray 210 are equivalent to the size of the slide observation support of the ImageXpress Pico machine;

[0040] Figure 13. Views of MG63 spheroids obtained in the device 100 and in a standard multi-well plate (96 well plate) have a comparable morphology. The Figure shows representative transmitted light images of MG63 cell spheroids acquired daily up to 4 days (24, 48, 72 and 96 hours after seeding). The row of images above is related to the MG63 cells seeded in the agarose platform, at the selected concentration of 5,000 cells / microwell. The row of images below is for MG63 cells seeded in the wells of a standard U-bottomed multi-well plate (image 96 well plate), at the selected concentration of 5,000 cells / well (10x magnification, representative images). At the macroscopic and qualitative level, it appears that the spheroids obtained in the agarose platform have a morphology comparable to the spheroids obtained in standard support; the scale bar, at the bottom right of each box, corresponds to a length of 200 pm;

[0041] Figure 14. Views of spheroids of 143B obtained in device 100 according to the present invention and in a standard multi-well plate. Representative transmitted light images of 143B cell spheroids acquired daily up to 4 days (24, 48, 72 and 96 hours after seeding). The row of images below is for 143B cells seeded in an agarose device 100, at the selected concentration of 5,000 cells / microwell. The row of images above is for 143B cells seeded in the wells of a standard U-bottomed multi-well plate (in the image, 96 well plates), at the selected concentration of 5,000 cells / well (10x magnification, representative images). At the macroscopic and qualitative level, it appears that the spheroids obtained in device 100 have a morphology comparable to the spheroids obtained in this standard multiwell plate; the scale bar, at the bottom right of each box, corresponds to a length of 200 pm.

[0042] Figure 15. Graphs of spheroid diameters of MG63 and 143B cells (shown in Figures 13 and 14) obtained by manual quantification (n = 30 spheroids, maximum and minimum diameter for each spheroid, mean ± SEM, uncoupled two-tailed t-test, * p <0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 compared to 24 hours, ###p<0.001 and ####p<0.0001 spheroids obtained in the agarose device 100 compared to spheroids obtained in the standard plate at 96 wells - 96 well plate);

[0043] Figure 16. Images of a row of MG63 and 143B cell spheroids in the agarose device 100, acquired using an ImageXpress Pico automated image acquisition microscope (4x magnification, 1 mm scale bar). Spheroids, from both lines, were kept in culture for 48 hours in agarose device 100. Prior to acquisition, the spheroids were labeled with a fluorescent vital dye (Calcein AM). Calcein AM allows to see both the morphology of the spheroid and to have qualitative information on the viability of the cells in the outer part of the spheroid; Figure 17. Representative images of a spheroid of MG63 cells and one of 143B cells stained with live / dead kits placed in a device 100 according to the present invention and acquired by double photon confocal microscope, volume render, NIR Apo objective 40x immersion, numerical aperture 0.8, refractive index 1.333, resonant scanning, multi photon confocal laser scanning, zoom at 1 , z-step 2 pm, 108 Zstack loops with Ni-E ZDrive (Nikon). For excitation, a laser power of 21 .1 , a laser wavelength of 820 was used;

[0044] Figure 18. Representative images of spheroids of MG63 treated with increasing doses of doxorubicin (0 pM, 2.5 pM, 5 pM, 10 pM) for 3 days in a device 100 according to the present invention. The images were acquired with an automated imaging fluorescence microscope. Prior to acquisition, the spheroids were labeled with Calcein AM, a vital dye. Qualitatively, it appears that the morphology of the spheroids is not affected by treatment with doxorubicin. The morphology of MG63 spheroids is not affected by treatment with increasing doses of doxorubicin;

[0045] Figure 19. Representative images of spheroids of 143B treated with increasing doses of doxorubicin (0 pM, 2.5 pM, 5 pM, 10 pM) for 3 days in a device 100 according to the present invention. The morphology of 143B spheroids appears to be influenced in a dose-dependent manner by treatment with increasing doses of doxorubicin. The images were acquired with an automated imaging fluorescence microscope. Prior to acquisition, the spheroids were labeled with Calcein AM, a vital dye. Qualitatively, it appears that the morphology of the spheroids changes;

[0046] Figure 20. Graph showing the trend over time of the size of MG63 spheroids, expressed as a percentage of untreated spheroids at time 0 (TO), when treated with increasing dosage of DXR (0 pM, 2.5 pM, 5 pM, 10 pM) in a device 100 according to the present invention. The size of MG63 spheroids treated at increasing dosages of doxorubicin does not have a dose-dependent trend. For the analysis of the size differences of the spheroids (treated vs untreated at TO) we used the t-test (* # § refer respectively to 2.5 pM, 5 pM, 10 pM respectively, “ p < 0.01 , **** p < 0.0001 , ### p < 0.001 , #### p < 0.0001 , §§§§ p < 0.0001 vs spheroids before treatment at TO);

[0047] Figure 21. Graph showing the trend over time of spheroid size expressed as a percentage of untreated spheroids at time 0 of 143B, when treated with increasing dosage of DXR (0 pM, 2.5 pM, 5 pM, 10 pM) in a device 100 according to the present invention. For the analysis of spheroid size differences (treated vs. untreated at TO) we used the t-test (* # § are referred respectively to 2.5 pM, 5 pM, 10 pM respectively, ** p < 0.01 , **** p < 0.0001 , ## p < 0.01 , #### p < 0.0001 , §§ p < 0.01 , §§§§ p < 0.0001 vs spheroids before treatment at TO). The size of 143B spheroids treated at increasing dosages of doxorubicin shows a dose-dependent trend;

[0048] Figure 22. The figure shows a comparison chart between the Alamar Blue test and the AP test for drug screening with the agarose platform. The figure shows a graph showing viability values of the spheroids MG63 (top) and 143B (bottom) obtained with two indirect tests in a device 100 according to the present invention. The values are expressed as a percentage of the untreated samples, at the same time point. Spheroids were treated with increasing dosage of doxorubicin (0 pm, 2.5 pm, 5 pm, 10 pm). The Alamar Blue test and the AP test were performed 72 hours after treatment (Mann-Whitney two-tailed unpaired, *p < 0.05 and ****p < 0.0001 vs 0 pM DXR; #p < 0.5 vs AP test, ##p < 0.01 compared to AP test).

[0049] In the attached figures to the same numbers correspond equal elements.

[0050] Definitions

[0051] In the context of the present invention, reference is made to quantities and elements for which a definition is provided below within the scope of the invention.

[0052] • Major axis (of an element): geometric axis directed parallel to the spatial dimension (height, width or length) of greatest extension of an element and passing through the geometric center (in three- dimensional space) of said element;

[0053] Chamfer and its bending radius: in the present invention the term "chamfer" means a rounded fillet between several surfaces (or on a single surface in the case of the chamfer on the vertex of a cone) that has a certain "bending radius" defined as the radius of the arc of circumference that is obtained by sectioning said chamfer with any plane containing the major axis of the chamfered geometric figure;

[0054] In this document, the term "w / v" refers to a mass / volume ratio, i.e. the percentage ratio of grams of solute per 100 ml of solution);

[0055] According to the present invention, the term "biopsy tissue graft" refers to the collection of a tissue sample taken from a patient via a biopsy in the context of a laboratory study and subsequently implanted or cultured in vitro for research aims;

[0056] According to the present invention, the terminology "patient-derived tissue (PDT)" or patient-derived tissues refers to tissue samples taken directly from human patients, usually by biopsy or surgery;

[0057] According to the present invention, the term "ECM" refers to the extracellular matrix. The extracellular matrix is a three-dimensional structure that is located outside cells and is made up of protein and non-protein components that provide structural support and regulate communication between cells;

[0058] According to the present invention, the operation of "thermal crosslinking" of agarose solution is referred to as a process by which a solution, e.g., agarose solution, changes from a sol state to a gel state. The process is temperature-induced. Between 90°C and 100°C, agarose is in a sol (liquid) state. By progressively decreasing the temperature, the polymer chains of agarose become immobilized and form bonds between them, and the solution passes to a gel state (semi-solid gelatinous state); • In this document, the terms "approximately" or "around" as used herein when referring to a measurable value such as a quantity, a time duration, and the like, are intended to encompass variations of ±20%, ±10%, ±5%, ±1 %, or ±0.1 % from the specified value, wherein such variations are appropriate to perform the methods described.

[0059] Detailed description of the invention

[0060] The device for obtaining and observing three-dimensional samples according to the present invention makes it possible to overcome the problems and limitations highlighted in known art. The operative part of the present invention makes it possible to:

[0061] - perform assays on 3D cell samples / models for time-lapse or confocal microscopic observations, keeping the sample immobilized and viable (if necessary) for the duration of the observation, and avoiding causing damage or structural alterations to the sample itself;

[0062] - The device, according to the present invention, also allows to observe the samples under analysis with the techniques mentioned above during the performance of biological assays for pharmacological screening or for the identification of new therapeutic targets;

[0063] - depending on the required needs, the device according to the present invention can be made with microstructures (i.e. cavities and compartments) that can be customized and adaptable to different tumor forms (for instance with a "V" structure for the formation and housing of spheroids, or a "U" structure for housing PDTs and other cellularized 3D constructs, such as bioprints);

[0064] - another advantage of the device according to the present invention is that it can observe the fluorescent signals and locate them in the 3D sample (also on the Z axis, i.e. the axis relative to the depth of the device as shown in FIG. 1 1 ) through the use of immersion objectives. This therefore allows a direct observation with a higher optical resolution (compared to observation with a reversed / inverted microscope) of the still viable sample, and provides much more information to the observer than current devices;

[0065] - the device, according to the present invention, also allows to obtain cell spheroids of both micro and millimeter dimensions so as to have cell models that are more representative and resemble the patient's pathological tissue. Spheroids can be made directly inside the invention device with techniques known to the expert in the field. The device can in fact be used both for obtaining and for observing cellular spheroids. One or more cellular spheroids are obtained with the device of the present invention by seeding cells inside one or more cavities of the device, said cells, by gravity, are deposited at the bottom of the cavities assembling themselves to form a spheroid. In this way, with the device of the present invention, intermediate manipulations between obtaining the spheroids and their observation are avoided, and it is indeed possible to observe the process of formation of said spheroids. Specifically, the spheroids that can be obtained with said device are preferably between 500 pm (10-6m) and 3 mm (10-3m);

[0066] - combine factors such as three-dimensionality, cellular heterogeneity (e.g. endothelial cells to simulate tumor vascularization), ECM, the creation of gradients of metabolites and nutrients, oxygen and pH, as in the in vivo tissue, essential for a good level of prediction of the patient's response to drug therapy. The predictivity of therapeutic efficacy compared to the models and analysis processes currently available in known art is therefore increased;

[0067] - it can be used with microscopes of various sizes and can also be mounted on a motorized stage used for Time-Lapse observations, having dimensions in plan approximately equal to those of histology slides (preferably about 75 mm x 25 mm, or 3 in x 1 in); given these dimensions, the device is compatible for analysis with conventional manual, but also semi-automated or automated, microscopes making the developed technology accessible to many more future users and reducing acquisition times.

[0068] The process of making the device involves the use of low-cost, easily available, accessible and easy-to-use equipment, technologies and materials, e.g. a 3D printer. This process is also potentially scalable and feasible in all biological laboratories, without requiring specific engineering skills.

[0069] The device according to the present invention is designed to allow the development of spheroids (or other organoids) on the device itself, avoiding the damage of the cell models and / or the formation of artifacts during their transfer from a support wherein they have been developed (common practice in the state of the art) to the one wherein they are to be observed. The 3D constructs obtained, for instance spheroids, can then be made inside the device housings and then directly observed and analyzed, intact and viable, by means of microscopy techniques, in timelapse or confocal mode of 3D samples, both suspended in the culture medium and fixed.

[0070] In addition, for the execution of biological assays, the device according to the present invention requires much smaller volumetric quantities than those required or necessary for standard platforms.

[0071] In order to allow experts in the field to better understand the technical solutions of this invention, some non-limiting embodiments will be described in detail below, also with reference to the attached drawings.

[0072] The elements and characteristics illustrated in the various preferred embodiments, comprising drawings, can be combined with each other without leaving the scope of protection of the present invention as described below.

[0073] Description of a device implementation

[0074] With reference to FIG. 1 , a top view of a device 100 according to the present invention is shown. In FIG.1 , as in the description below, by way of example and not limitation, the embodiment of the present invention considered the best to date is shown.

[0075] Device 100 of the present invention is designed to allow direct biological assays to be conducted using 3D three-dimensional models immersed in cell culture medium, preferably in phosphate-buffered saline solution (PBS).

[0076] Advantageously, said device 100 comprises one or more rows of cavities. 101.

[0077] Preferably but not limited to, said device 100 comprises three rows of cavities 101 .

[0078] Said rows comprise a number of cavities 101 preferably between 1 and 30, preferably between 10 and 25, optimally between 15 and 22.

[0079] Said cavities 101 can have, by way of example and not limited to conical, conical blunted shape, truncated conical bevel, hemispherical, cylindrical and / or parallelepipedal shape with a polygonal base, shapes with irregular depth (Z axis - as shown in FIG. 1 1 ) (adaptable to standard non-geometric shapes, such as those of cell scaffolds), or other type of cavity suitable for housing the biological material to be examined.

[0080] With reference to FIG. 1 , a device 100 is shown, comprising three rows each with twenty-one cavities 101 of a blunted conical shape.

[0081] Said cavities 101 having a depth within the thickness of the device 100 preferably between 20% and 80% of said thickness.

[0082] Advantageously, said rows of cavities 101 further comprise a compartment 102 from which these cavities 101 depart, within the thickness of the device 100. This compartment 102 preferably has the shape of a parallelepipedal with a rectangular / square section with the major axis oriented parallel to the major axis of said device 100.

[0083] According to a preferred but not limiting embodiment of the present invention, said cavities 101 have a substantially conical shape with a chamfer at the vertex (distal to the circular base of the cone and placed deeper within the thickness of said device 100) with a bending radius preferably between 1 % and 10% of the diameter of the circular base of the cone and ideally equal to 5%.

[0084] According to a preferred but not limiting embodiment of the present invention, said cavities 101 have a truncated-conical shape with a larger base in contact with the outer edge of the device and / or with said compartment 102.

[0085] Said truncated-conical shape can have an inclination of the side wall with respect to said major base, preferably between 85° and 60°.

[0086] Preferably, the values of the inclination angles are 85° and 75° with respect to said major base.

[0087] Said truncated-conical shape has a smaller base and / or a circular chamfer. This minor base is not present when the bending radius of said circular chamfer is greater than or equal to the radius of said minor base. This minor base is connected to the side wall of said truncated-conical shape through said chamfer with a bending radius preferably between 1% and 45% of the diameter of the major base.

[0088] Preferred values of this bending radius are the values equal to 5%, 25% and 45% of the diameter of said major base.

[0089] Said cavity 101 has a depth preferably between 80% and 200% of the maximum size of its base (for instance the diameter in the case of conical, blunted conical, chamfered truncated conical, cylindrical, semi-spherical cavities), more preferably between 90% and 150% and optimally equal to 100%.

[0090] Said compartment 102 has a depth preferably between 80% and 200% of the depth of said cavity 101 , more preferably between 90% and 150% and ideally equal to 100%.

[0091] Said cavity 101 has a base with a maximum dimension (e.g. the diameter in the case of conical, conical chamfered, chamfered truncated conical, cylindrical, semi-spherical cavities) between 5% and 20% of the smaller size in plan (width vs length) of said device 100.

[0092] With reference to FIG. 4, a section of a device 100 is shown with three rows of truncated-conical cavities 101 with a side wall having an inclination of 75° with respect to the largest base; said FIG. 4 also shows three rows of compartments 102. Said FIG. 4 shows a first cavity 101 ' which has a chamfer 1 1 ' with a bending radius equal to 30% of the diameter of the major base, a second cavity 101 " which has a chamfer 1 1 " with a bending radius equal to 25% of the diameter of said major base and a third cavity 101 '" which has a chamfer 1 1"’ with a bending radius equal to 5% of the diameter of said major base.

[0093] With reference to FIG. 5, a section of a device 100 is shown with three rows of truncated-conical cavities 101 with a side wall having an inclination of 85° with respect to the largest base.

[0094] Said FIG. 5 also shows three rows of compartments 102.

[0095] In said FIG. 5 is shown a first cavity 101 a which has a chamfer 11 a with a bending radius equal to 45% of the diameter of the major base, a second cavity 101 b which has a chamfer 1 1 b with a bending radius equal to 25% of the diameter of said major base and a third cavity 101 c which has a chamfer 1 1 c with a bending radius equal to 5% of the diameter of said major base.

[0096] Preferably, in some embodiments of the present invention, said device 100 has rounded edges.

[0097] Preferably, in certain non-limiting embodiments of the present invention, said device 100 has plan dimensions of between 50 mm and 200 mm in length and between 15 mm and 50 mm in width.

[0098] Said dimensions in plan even more preferable are about 75 mm x 25 mm.

[0099] These dimensions in plan being, in some embodiments of the invention, preferably equal to about 68 mm x 18 mm.

[0100] Said device 100 has a thickness preferably between 5 mm and 30 mm and even more preferably equal to about 6.50 mm.

[0101] Said device 100 being preferably obtained by injection of a solution of 2% w / v agarose into a mould 200.

[0102] Said mould 200 (shown in FIG. 7) comprising a tray 210 and a cap 220. With reference to FIG. 2 and 3, two top views are shown respectively of said tray 210 and said cap 220.

[0103] Said tray 210 being provided with a compartment or recess 21 1 of dimensions equal to the width, length and overall thickness of the device 100 to be obtained through the forming process, detailed below.

[0104] Said cap 220 suitable for being placed on said tray 210 occupying part of said compartment 211 .

[0105] Said cap 220 is shaped in such a way as to comprise male surfaces on which a plurality of projections or overhangs 221 are obtained that determine corresponding female shapes, said projections 221 being insertable within said female surfaces generically made in the form of a compartment and indicated by 101 , wherein the male surface is configured to be inserted inside said female surfaces to fill the spaces within said space or compartment 21 1 . Those cavities 101 and compartments 102 of said device 100 are generated by means of a forming process described below. Specifically, the projections 221 have, therefore, the shape and dimensions to create these cavities 101 , the shapes can be chosen, by way of example but not limitation, between: conical, blunted conical, blunted truncated-conical, hemispherical, cylindrical and / or parallelepipedal with a polygonal base. The shapes and their dimensions can be chosen in the most appropriate way by the expert in the field who reads the information contained in this description and in the light of his or her own technical knowledge.

[0106] A section of a cap 220 with three rows of blunted conical projections is shown with reference to FIG. 6A. FIG. 6B shows the corresponding female section of a device 100 obtained with said cap 220 shown in FIG. 6A. In said FIG. 6B these cavities 101 have a chamfer 1 1 with a bending radius equal to about 5% of the diameter of said major base, corresponding to the chamfer shown in FIG. 6A on said projections 221 .

[0107] Said cap 220 preferably comprising one or more holes 222 for the injection of the polymer (which may be a solution of 2% w / v agarose) into mould 200, within said compartment 211 , when said cap 220 is placed on said tray 210. Said holes 222 preferably have a circular cross-section and a diameter preferably between 1 mm and 5 mm and even more preferably equal to about 3 mm.

[0108] Preferably, said mould 200 can be made through a 3D printing process using the Fused Deposition Modeling technique, using a commercial PLA (polylactic acid) filament.

[0109] Said tray 210 of said mould 200 can also be made with commercial polydimethylsiloxane (PDMS) wherein the PDMS is prepared by mixing with a curing agent [Sylgard 184, Dow Corning Corp., Midland, Ml, USA] at a ratio of 10:1 w / w, with known techniques, for instance by pouring it onto a master mould. After the 24-hour resting period at 37°C, it is possible to remove said tray 210 from said master mould.

[0110] In other non-limiting embodiments of the present invention, said mould 200 is made with light-curing epoxy / phenolic resins (synthetic polymers). Printing can take place through a stereolithographic printing process (which has a higher accuracy than continuous wire 3D printing) wherein these epoxy / phenolic resins polymerize through the action of UV rays.

[0111] The printing methods of the mould 200 are conventional and the polymeric materials that can be used are also conventional.

[0112] For instance, tray 210 can be made of any rigid or semi-rigid polymeric material, as long as it is transparent to microscopic observation. Synthetic polymeric materials that can be used are, for instance, polydimethylsiloxane (PDMS), polyethylene glycol (PEG), polyacrylamide, polymethylmethacrylate (PMMA), polystyrene, polycarbonate, polyurethane. Glasses and ceramics, such as borosilicate glass, bioceramics, and amorphous silica, can also be used, but they can be more brittle than polymers.

[0113] The cap 220 can also be made of any polymer material and with any forming process. Commercial polylactic acid and traditional 3D printing are preferred, but other commercial polymeric materials can also be used, such as polycaprolactone (PCL), polyoxymethylene (POM), polyetheretherketone (PEEK), thermoplastic polyurethane (TPU). In addition to these, there are also other emerging materials such as polylactate-co-glycolate (PLGA), polycarbonate (PC), and polyhydroxybutyrate (PHB). and also different moulding techniques such as the technique known as fused deposition modeling (FDM), stereolithography (SLA), Digital Light Processing (DLP), PolyJet.

[0114] The tray 210 and the cap 220 constitute the mould and counter-mould of the device 100 according to the invention which can be made with a polymer that has the following characteristics: to be a hydrogel. Hydrogels are water-insoluble polymer networks organized in 3D structures, capable of absorbing large quantities of water or biological fluids thanks to the presence of hydrophilic functional groups in their structure. Hydrogels are divided into two categories depending on the type of polymer they are made of: natural and synthetic hydrogels. In turn, hydrogels of natural origin can be made up of polysaccharides (such as chitosan, cellulose, alginate or agarose) or protein (such as collagen or gelatine). to keep gelling in the range of 0-40°C; to be biocompatible, i.e. to have (Consensus conference on biomaterials, Chester, UK - 1986) the ability (for a material used for a given application) to act by determining an appropriate response in the host. In this specific case, the material is biocompatible because it must be able to support the proliferation and normal functioning of cells, without inducing cytotoxicity or inflammatory responses; to be repellent to cells and test samples, i.e. possess characteristics that make it unfavourable to cell adhesion. This "repellent" characteristic is often exploited in biological research, for instance to keep cells suspended in culture or to study cell adhesion and behaviour on unfavourable surfaces. A particularly suitable polymer is agarose, e.g. commercial 2% agarose. Agarose is a hydrogel of natural origin, biocompatible, and repellent to cell adhesion. Agarose is a highly hydrophilic hydrogel, which means it has a strong affinity for water molecules. This characteristic creates an aqueous environment that does not favour cell adhesion, as cells tend to prefer more hydrophobic surfaces to anchor themselves. Unlike many other hydrogel matrices, agarose does not contain binding sites for cell adhesion proteins, such as arginine-glycine-aspartic acid (RGD). Without these anchor sites, cells find it difficult to adhere and proliferate on the surface of agarose. The highly ordered reticular structure of agarose creates an environment that is physically unfavourable to cell adhesion. Cells prefer more disordered and porous environments in order to integrate and spread.

[0115] Production method

[0116] A method of manufacturing such a device 100 preferably comprises the following steps: forming the two elements of the mould 200 separately, e.g. by 3D printing tray 210 and cap 220 of said mould 200. The tray 210 is manufacturable in PDMS, the cap 220 is manufacturable in PLA; positioning said cap 220 on said tray 210 in such a way that said projection 221 engages said compartment 211 ; injecting said 2% w / v agarose solution into compartment 21 1 , e.g. through a nozzle inserted into a hole 222; other biocompatible polymers with cell-repellent properties are for instance PEG, Polyvinylpyrrolidone (PVP), hyaluronic acid, polymethylmethacrylate. However, these polymers do not have the same type of cross-linking as agarose. Said polymers cannot be injected through the holes in the cap and then cross-linked with the temperature to make them take on a defined shape, i.e. the negative of the cap structure. For this reason, agarose is the most suitable polymer for a device that has these characteristics. thermal cross-linking of the injected agarose solution, at a temperature between 0°C and below 40°C; preferably between 0°C and 12°C, preferably equal to about +4°C and for a time preferably between 15 and 30 minutes and even more preferably equal to about 20 minutes; when said agarose solution turns out to be a formed body (reaching the end of a gelation process), i.e. substantially easy to handle, it generates said device 100; removing said cap 220.

[0117] The device 100 can also be used by leaving it inside said tray 210.

[0118] In some embodiments of the invention, device 100 is used without said tray 210, in such embodiments, said method of production comprises a final stage of removal of device 100 from said tray 210.

[0119] With reference to FIG. 7, some steps of a method of production of a device 100 according to the present invention are shown.

[0120] The first image at the top in FIG. 7 shows the positioning step of said cap 220 on said tray 210, wherein the arrow shows the direction of support of the cap 220 on the tray 210. The second image at the top in FIG. 7 shows the injection step of said 2% w / v agarose solution through a nozzle inserted into said hole 222.

[0121] The third image at the top in FIG. 7 shows the removal step of cap 220 when said device 100 has been obtained by thermal cross-linking of the injected agarose solution.

[0122] The image at the bottom in FIG. 7 shows said device 100 extracted from said tray 210.

[0123] Advantageously, said mould 200 can be used for the production of a plurality of devices 100 through these injection, thermal cross-linking and removal steps of the production method.

[0124] The invention has been described with reference to exemplary embodiments that are not to be considered limiting the scope of the requested protection. What is illustrated in the figures is merely illustrative and a person experienced in the field can make variations or modifications that do not depart from the spirit and scope of the invention. It is understood that such variations and modifications are comprised in the scope of the description and claims.

[0125] Device Applications The device of the present invention described above can be used by way of example but not limited to for applications such as:

[0126] • cultivation and achievement of spheroids, as 3D tumor models for preclinical studies;

[0127] • microscopic observations (e.g., viability assays and fluorescence / immunofluorescence stains) of three-dimensional experimental models, comprising spheroids / organoids, cellularized scaffolds of different nature and shape, bioprints, or biopsy grafts from patient tumor;

[0128] • housing and subsequent characterization of already formed spheroids;

[0129] • execution of biological tests, stains, immunofluorescence;

[0130] • observations of cellular phenomena;

[0131] • preclinical drug screening and drug development.

[0132] In particular, device 100 of the invention allows the creation of patient- derived tissue (PDT) cultures, grafts, bioprints, cellularized scaffolds or spheroids, also obtained from cells disrupted from the patient's tumor. This makes it possible to carry out preclinical research with high predictive potential for the study of cellular behaviour, expression and localization of molecules, or for in vitro screening of drug efficacy.

[0133] Thanks to the improved in vitro predictivity of drug efficacy, there is also the possibility of reducing the number of animals to be used as a preclinical model for drug screening.

[0134] It is also possible to identify the experimental therapeutic protocol to be used in the specific patient, thanks to the pre-screening of the drugs tested on the 3D cellular model developed from the patient's cells, according to a precision medicine approach.

[0135] Cell culture

[0136] Before their use to form spheroids in the platform, the cells are kept in culture in standard two-dimensional supports (e.g. Flask, Tissue Culture Flask #ET7076) in IMDM medium, with supplemented 10% FBS and 1 % pen / strep. At the time of seeding, the cells are detached from the Flask using a Trypsin / EDTA solution and counted, according to standard methods. A cell suspension is then prepared to take into account the number of cells per well, the number of wells per row (n = 20) and the volume for each row (V = 200 pL). For instance, three seeding densities were tested: 2,500 cells / well, 5,000 cells / well, 7,500 cells / well. Considering the individual rows of the device, three cell suspensions were respectively prepared as follows: 50,000 cells / 200 pL (2,500 cells * 20 wells), 100,000 cells / 200 pL (5,000 cells * 20 wells), 150,000 cells / pL (7,500 cells * 20 wells). The cell suspension is seeded "dropwise" on the row of the platform. In the first 6 hours after seeding, the cells are evenly distributed in the wells. Within 24 hours, the cells aggregate to form spheroids. The change of medium is done every two days. Spheroids can be kept in culture for up to at least 10 days, in an incubator (37°C, humidity 95%, CO25%).

[0137] Examples

[0138] The embodiments shown below are to be considered as merely illustrative and not limiting the scope of the present invention.

[0139] Instrumentation and reagents used

[0140] Scanner ImageXpress Pico Milteny + Texas Red Filter Cube (14532-1 )

[0141] Nikon dual photon spectral confocal microscope (A1 R MP confocal microscope, Nikon, Tokyo, Japan)

[0142] Nikon ECLIPSE inverted microscope - TS100 Stereomicroscope SM Z 18 Zoom Body Nikon+ DS-Ri2 Digital Microscope camera (#700349)

[0143] Aura Mini Vertical Laminar Flow Hood (#K3839, BIOAIR INSTRUMENTS, Italy)

[0144] Culture medium: IMDM (#42200, Life Technologies, Carlsbad, CA, USA)

[0145] Penicillin (20 U / mL), streptomycin (100 mg / mL) (#ECB3001 D), Euroclone, Italy) (pen / strep)

[0146] Fetal bovine serum (#ECS5000L Euroclone, Italy) (FBS)

[0147] Trypsin 0,05%- EDTA 0,02% in PBS 100ml (#ECB3052D, Euroclone, Italy)

[0148] Doxorubicin hydrochloride (# d1515, Sigma-Aldrich, USA) Agarose (#50100E, Antibiotic Products Company, Italy) Calcein AM (#c3099, Molecular Probes USA)

[0149] Live / Dead cell imaging kit 488 / 570 (#R37601 , Invitrogen USA) Alamar Blue (#DAL1100, Invitrogen, USA)

[0150] 4-nitrophenil phosphate disodium salt hexahydrate (tablet) (test AP) (#N2765, Sigma-Aldrich, USA).

[0151] Example 1

[0152] In an implementation example of the present invention, the device was used for the development and characterisation of homotypic osteosarcoma spheroids, from two commercial lines 143B (ATCC, #CRL- 8303) and MG63 (ATCC, #CRL-1427) (figure 8 and figure 9). Osteosarcoma is a very aggressive primary bone cancer that occurs in children and adolescents, with a strong tendency to metastasize and not responding to current therapies.

[0153] Following the method described below, an agarose device was created with a cavity surface and dimensions comparable to those of an imaging slide. (FIG. 9 and FIG 10)

[0154] Preparation of the agarose device

[0155] The components necessary to obtain the agarose device, specifically, said tray 210 and said cap 220, were designed with the help of the Autodesk Inventor 3D modeling software.

[0156] Tray 210 and cap 220 were then 3D printed using the Fused Deposition Modeling technique, using a commercial filament of PLA (polylactic acid) for cap 220, and using PDMS for tray 210. The plan dimensions in the x, y plane of the PDMS tray 210 are 25 mm (width) and 75 mm (length) equal to those of a standard imaging slide (FIG. 10). Subsequently, with the help of a structurally incorporated groove, cap 220 was placed on the surface of PDMS tray 210.

[0157] Once tray 210 and cap 220 were assembled, a solution of agarose 2% w / v was inserted into tray 210 in PDMS through a first hole 222 on cap 220 until it was completely filled, which can be deduced from the leakage of the solution from a second hole 222 of said cap 220.

[0158] Once the filling was complete, the agarose was thermally cross-linked (process wherein the polymer chains crystallize) at +4°C for 20 minutes and the subsequent removal of cap 220 provided the device 100 (FIG. 8, 9, 10). Thermal cross-linking is a process by which an agarose solution changes from a sol (liquid) state to a gel (gelatinous) state. The process is temperature-induced. Between 90 and 100 degrees, agarose is in a sol (liquid) state. By progressively decreasing the temperature, the polymer chains of agarose become immobilized and form bonds with each other, and the solution changes to a gel state (semi-solid gelatinous state). This process does not require the use of cross-linking agents.

[0159] Device 100 is "microstructured" with a microstructuring consisting of 3 parallel rows of 20 cone-shaped cavities 101 (wells) (height 2 mm, surface diameter 2 mm) (as shown in FIG. 8, 9, 10).

[0160] The possibility of housing the device 100 in a slide insert of the A1 R MP confocal microscope (Nikon, Tokyo, Japan)) (FIG. 1 1 ) was also successfully evaluated.

[0161] The possibility of keeping the spheroids immersed and viable in complete IMDM medium, with the addition of FBS 10% and pen / strep 1%) while being observed, using both air and water objectives was also verified (FIG. 13 and 14).

[0162] The possibility of acquiring images in an automated way with the ImageXpress Pico - Automated Cell Imaging System microscope by housing the device in the sample holder of the instrument was then successfully verified. As can be seen from figure 12, the ImageXpress Pico microscope recognizes the device 100 and automatically acquires the three rows of fluorescence-labeled spheroids (n = 60 total spheroids) (Calcein AM, vital dye). The acquisition, with a 1 mm Z stack, takes place in a time of about 20 minutes. This makes it possible to speed up the acquisition of a large number of spheroids and to obtain qualitative (e.g. morphology) and quantitative (diameter) information in a short time.

[0163] The morphology of the spheroids was assessed by acquiring images with the ImageXpress Pico tool. Spheroids were seeded and grown in the device from two osteosarcoma cell lines (143B and MG63, ATCC #CRL8303 and #CRL1427) 72 hours after seeding the spheroids were fluorescence-labeled with a vital dye (Calcein AM). Figure 8D shows spherical spheroids and all having the same morphology. Wherein by "all having the same morphology" we mean that, keeping the parameters for obtaining spheroids unchanged, the process leads to the obtaining of spheroids always with the same shapes and sizes. The difference in compactness detected in spheroids depended on the cell line used. In fact, spheroids formed by MG63 cells are structurally more compact due to the secretion of extracellular matrix. (FIG. 16).

[0164] The morphology of the spheroids was further investigated by acquiring images with the A1 R MP confocal microscope (Fig.17). The confocal microscope combined with an immersion objective allows to observe the spheroid in detail, both at the surface level and inside it, generating high- resolution images. Vital staining was also carried out to demonstrate the possibility of visualising the living spheroid.

[0165] Example 2

[0166] In another embodiment, an evaluation of the dimensions of the spheroids obtained with the device 100 according to the present invention was carried out, comparing them with those of the spheroids obtained through commercial platforms (FIG. 15).

[0167] As shown in figure 15, it has been shown that the trend over time of the size of homotypic spheroids of 143B obtained by means of a agarose device 100 is comparable to that of spheroids obtained with a standard technique known to the expert in the field (U-bottom 96-well plate, control FIG. 14, 15), with the same number of cells. The tests were repeated for 10 days, trying different seeding densities (2,500 cells / well, 5,000 cells / well, 10,000 cells / well). Every 24 hours, images were acquired using an inverted brightfield microscope (10x magnification) and the images were subsequently reprocessed using the Imaged image processing software.

[0168] The trend in the size of homotypic spheroids of MG63 obtained by agarose device was then monitored over time. This trend, as can be seen from figures 13 and 15, was comparable to that of spheroids obtained with a standard technique known to the expert in the field (U-bottom 96-well plate), with the same number of cells. The test was carried out for up to 10 days, trying different seeding densities (5,000 cells / well, 10,000 cells / well). Every 24 hours, images were acquired using an inverted brightfield microscope (10x magnification) and the images were subsequently reprocessed using the Imaged image processing software.

[0169] On the basis of the results, the seeding density of 5,000 cells / well was selected to proceed with a further characterization of the spheroids, both at the morphological (diameters, FIG. 15) and biological level. Specifically, the device was found to be suitable for performing indirect viability assays (Alamar Blue assay and AP test) on spheroids. Spheroids obtained from both lines were found to be vital.

[0170] Example 3

[0171] In another embodiment according to the present invention, the device 100 was evaluated suitable for drug screening.

[0172] The homotypic spheroids of 143B and MG63 were treated with doxorubicin (DXR), a standard drug indicated for the treatment of osteosarcoma, and the effect of doxorubicin at increasing dosages on the trend over time of spheroid diameters (Fig. 20 and 21 ), morphology (Fig. 18 and 19) and viability (Fig. 22) was evaluated.

[0173] For the evaluation of diameters, images were acquired and processed (Imaged) by means of an inverted microscope every 24 hours and it was seen that the spheroids of 143B, 72 hours after the administration of the DXR, show a logarithmic dose-response trend. The morphology, on the other hand, was evaluated by acquiring images using ImageXpress Pico (Fig. 18 and 19). Viability was assessed by two indirect assays: Alamar blue (22) and acid phosphatase assay (Fig. 22). With both tests, the logarithmic dose-response trend of the 143B spheroids was confirmed.

Claims

CLAIMS1. A device (100) made of solidified agarose gel, comprising one or more rows of cavities (101 ); said device (100) being configured to be housed in a compartment (21 1 ) of a tray (210) and formed by moulding the agarose gel within a mould (200) comprising the tray (210) and a removable cap (220), said cap (220) having a plurality of projections (221 ) which generate the cavities (101 ) upon gel solidification, wherein the tray (210) and the cap (220) are made of a transparent plastic material.

2. The device (100), according to the previous claim 1 , wherein the cavities (101 ) have surfaces which are complementary to the surfaces of the projections (221 ) with a female-male combination.

3. The device (100), according to any one of the previous claims, wherein cavities (101 ) and, correspondingly, projections (221 ) have a shape chosen among: conical, blunted conical, blunted frusto- conical, hemispherical, cylindrical, parallelepipedal shape with a polygonal base, and cavities (101 ) are designed to house aliquots of the biological material to be examined.

4. The device (100), according to any one of the previous claims, wherein said rows of cavities (101 ) are housed in corresponding compartments (102) from which they depart, within the thickness of the device (100).

5. The device (100), according to any one of the previous claims, wherein the number of cavities (101 ) for each row is between 1 and 30, preferably between 10 and 25, more preferably between 15 and 22.

6. The device (100), according to any one of the previous claims, wherein said cavities (101 ) have a depth within the thickness of thedevice (100), preferably comprised between 20% and 80% of said thickness.

7. The device (100), according to any one of the previous claims, having plan dimensions with a length between 50 mm and 200 mm and a width of between 15 mm and 50 mm; said plan dimensions preferably being equal to about 75 mm x 25 mm, even more preferably equal to about 68 mm x 18 mm; the thickness of said device (100) being between 5 mm and 30 mm, more preferably equal to about 6.50 mm.

8. The device (100), according to any one of the previous claims, wherein said compartment (211 ) has equal dimensions to the width, length and overall thickness of the device (100) and wherein said cap (220) rests on said tray (210) engaging part of said compartment (21 1 ).

9. The device (100), according to any one of the previous claims, wherein one or more holes (222) are made in said cap (220); said holes (222) being suitable for receiving means for injecting a 2% w / v agarose solution in the mould (200), within said compartment (21 1 ), when said cap (220) is placed on said tray (210).10.The device (100), according to any one of the previous claims, suitable for:- housing biological material with dimensions ranging from millimeters to micrometers;- constituting a housing for obtaining spheroids of tumor cells;- housing 3D cellular constructs, spheroids, scaffolds, cellular structures, organoids and tissues; to allow automated microscopic visualization of biological material in suspension, in time-lapse, in inverted, confocal, fluorescence microscopy, with immersion objectives.

11. A process for the production of the device (100), according to anyone of the previous claims, wherein the tray (210) and the cap (220) constitute the mould and the counter-mould of the device (100); said process comprising the following steps: separately forming the tray (210) and the cap (220) which constitute the mould (200); positioning said cap (220) on said tray (210) ensuring that said projections (221 ) engage said compartment (21 1 ); preparing a 2% w / v agarose solution and injecting it inside the compartment (21 1 ), for instance through the holes (222); thermally crosslinking the injected agarose solution, at a temperature between 0°C and <40°C, preferably between 0°C and 12°C, preferably around +4°C and for a time sufficient for allowing the handling of the thus generated device (100), preferably comprised between 15 and 30 minutes and even more preferably equal to about 20 minutes; removing the cap (220).

12. The process, according to the previous claim 1 1 , wherein said mould (200) is made of a material chosen among: PLA (polylactic acid), polycaprolactone (PCL), polyoxymethylene (POM),Polyetheretherketone (PEEK), Thermoplastic polyurethane (TPU ), polylactate-co-glycolate (PLGA), polycarbonate (PC), polyhydroxybutyrate (PHB), and with a moulding technique chosen from: fused deposition modeling (FDM), stereolithography (SLA), Digital Light Processing (DLP), PolyJet, preferably the material being PLA and the technique being 3D printing.

13. The process, according to any one of the previous claims 1 1 -12, wherein said tray (210) of said mould (200) is made of a material chosen among: cross-linked polydimethylsiloxane (PDMS), polyethylene glycol (PEG), polyacrylamide, polymethylmethacrylate (PMMA), polystyrene, polycarbonate, polyurethane, glasses and ceramics, such as borosilicate glass,bioceramics and amorphous silica.

14. The process according to any one of the previous claims 1 1 -13, wherein said mould (200) is made with resins chosen between: photohardening phenolic epoxy resins or epoxy resins polymerized through the action of UV rays.

15. The process, according to any one of the previous claims 1 1 -14 wherein the cavities (101 ) take the shape of the projections (221 ) after the lifting of the cap (220) of the mould (200) from the tray (210), after the polymerized agarose solution has solidified.

Images