Rheo-optical method for the mechanical characterization of cellular spheroids and biopsies of biological tissues

Abstract

The present invention relates to a rheo-optical method for the mechanical characterization of cellular spheroids and biopsies of biological tissues through: • The use of samples without special preparations; • The execution of compression tests with a load (weight) within standard plates for cell cultures; • The possibility of applying the load (weight) using inexpensive and easily obtainable equipment; • The possibility of conducting the tests in a sterile environment; • The acquisition of images of the sample and their analysis for calculating the elastic and viscous modulus.

Description

[0001] RHEO-OPTICAL METHOD FOR THE MECHANICAL CHARACTERIZATION OF CELLULAR SPHEROIDS AND BIOPSIES OF BIOLOGICAL TISSUES

[0002] The present invention relates to a rheo-optical method for the mechanical characterization of cellular spheroids and biopsies of biological tissues.

[0003] STATE OF THE ART

[0004] The mechanical characterization of biological samples is the subject of intense research activity not only from a scientific perspective, but also in view of potential biomedical applications for diagnostic and therapeutic purposes. Biological samples of particular interest, in addition to tissue biopsies for pathological anatomy, are cellular spheroids, one of the most commonly used in vitro models of three-dimensional biology constructed in the laboratory. Cellular spheroids, which can be defined as cell aggregates formed in vitro, serve as a model of tissue microenvironment where cell-cell and cell-extracellular matrix (ECM) interactions are combined, present in numerous pathophysiological processes. Emerging applications of cellular spheroids are found in tissue engineering as constructs for implantable materials and in bioprinting as building blocks for complex three-dimensional structures. In all these applications, the mechanical behavior of cellular spheroids plays a key role in conjunction with biochemical factors in morphogenesis, tissue regeneration, and the development of pathologies.

[0005] Historically, interest in cellular spheroids has stemmed from the study of morphogenesis thanks to the pioneering work of Wilson (Wilson HV. On some phenomena of coalescence and regeneration in sponges. Journal of Experimental Zoology. 1907; 5:245-258), Holtfreter (Townes PL, Holtfreter J. Directed movements and selective adhesion of embryonic amphibian cells. Journal of Experimental Zoology. 1955; 128:53-120), and other authors (Shawky JH, Davidson LA. Tissue mechanics and adhesion during embryo development. Dev Biol. 2015 May l;401(l): 152-64), which inspired Steinberg and several scientists in developing the Differential Adhesion Hypothesis (DAH) (Steinberg MS. Reconstruction of tissues by dissociated cells. Some morphogenetic tissue movements and the sorting out of embryonic cells may have a common explanation. Science. 1963; 141 :401-408), based on the analogy with the phase separation of immiscible fluids, and the use of a tensiometer to measure the apparent tissue surface tension (ATST) (Foty RA, Forgacs G, Pfleger CM, Steinberg MS. Liquid properties of embryonic tissues: Measurement of interfacial tensions. Phys Rev Lett. 1994; 72:2298-2301). Subsequently, the DAH hypothesis was critiqued, leading to the proposal of the Differential Interfacial Tension Hypothesis (DITH) (Harris AK. Is cell sorting caused by differences in the work of intercellular adhesion? A critique of the Steinberg hypothesis. J Theor Biol. 1976; 61 :267-285., Brodland GW. The Differential Interfacial Tension Hypothesis (DITH): a comprehensive theory for the self-rearrangement of embryonic cells and tissues. J Biomech Eng. 2002 Apr; 124(2): 188-97). In most of these studies, cellular spheroids were compressed in a parallel plate apparatus, and stress relaxation (annealing) data were fitted to theoretical models to extract ATST and other mechanical parameters. The proposed concept in these studies is that following an initial elastic deformation, cellular rearrangement over longer timescales can be described in terms of ATST.

[0006] Experimental methods for characterizing the mechanical behavior of cellular spheroids include other techniques, such as isotropic compression via osmotic pressure (Dolega ME, Delarue M, Ingremeau F, Prost J, Delon A, Cappello G. Cell-like pressure sensors reveal increase of mechanical stress towards the core of multicellular spheroids under compression. Nat Commun. 2017 Jan 27;8: 14056) (Dolega ME, Monnier S, Brunel B, Joanny JF, Recho P, Cappello G. Extracellular matrix in multicellular aggregates acts as a pressure sensor controlling cell proliferation and motility. Elife. 2021 Mar 1 l;10:e63258.), Atomic Force Microscopy (AFM) (A-Hassan E, Heinz WF, Antonik MD, D'Costa NP, Nageswaran S, Schoenenberger CA, Hoh JH. Relative microelastic mapping of living cells by atomic force microscopy. Biophys J. 1998 Mar;74(3): 1564-78), cavitation rheology (Ruben C. Boot, Gijsje H. Koenderink & Pouyan E. Boukany (2021) Spheroid mechanics and implications for cell invasion, Advances in Physics: X, 6:1.), and elastography (Charles-Edouard Leroux, Joelle Palmier, Albert Claude Boccara, Giovanni Cappello and Sylvain Monnier, Elastography of multicellular aggregates submitted to osmo-mechanical stress, 2015, New J. Phys., 17, 073035). The application of these techniques has shown that the situation is made more complex by the potential fluid loss from the cellular spheroids, which occurs at relatively high values of imposed stress (this loss is about 15% at an imposed stress of 15 kPa and 17% at 40 kPa) and is likely due to the extrusion of water from the extracellular matrix. Comparable changes in cell volume occur at imposed stresses of 15-40 kPa. Moreover, the spatial distribution of stress within cellular spheroids is highly heterogeneous, with a softer and more viscous core (undergo a greater pressure) compared to the outer layer. The stress heterogeneity can be linked to the presence of a necrotic core in the cellular spheroid, caused by a lack of nutrients and the accumulation of waste products due to reduced diffusive transport at larger thicknesses. Mechanical properties also depend on the size and growth conditions of the cellular spheroids. Compression experiments between parallel plates and atomic force microscopy have been used to measure classical mechanical parameters, such as Young's modulus, and characteristic relaxation times, in both steady-state and transient conditions. While these techniques have provided significant scientific insights into the mechanical behavior of cellular spheroids, they rely on specialized equipment and technical expertise that are typically not available in cell biology laboratories.

[0007] Rheo-optical methods for the mechanical characterization of cellular spheroids and biopsies of biological tissues are also known, such as those presented in the publication by Li Hongliang et al., titled “Viscoelasticity Imaging of Biological Tissues and Single Cells Using Shear Wave Propagation,” published in Frontiers in Physics, vol. 9, 2021-06-21, XP093109774 (ISSN: 2294-424X, DOI: 10.3389 / fphy.2021.666192), and in the publication by Cheng Catherine et al., titled “Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses,” published in Journal of Visualized Experiments, no. I l l, 2016-05-03, pages 1-7, XP093110907 (ISSN: 1940-087X, DOI: 10.3791 / 53986).

[0008] For this reason, it is necessary to develop simple and more accessible experimental methods to measure the mechanical properties of cellular spheroids. Such methods should be compatible with standard cell culture techniques and relatively inexpensive, allowing the investigation of multiple samples in duplicate or triplicate, which is often required for the characterization of biological samples. The need to assay a large number of samples in a standardized manner to obtain experimental parameters that are easy to interpret is particularly relevant in the study of pathologies, where high variability in response is often encountered and tools are needed that allow for the analysis of a significant amount of data in a statistically acceptable manner.

[0009] BRIEF DESCRIPTION OF THE INVENTION

[0010] The invention relates to a rheo-optical method for calculating the elastic and viscous modulus of cellular spheroids and biopsies, comprising the following steps: a) inserting a sample to be analyzed into a well of a cell culture plate; b) applying a compressive force to the sample; c) acquiring single images or videos of the compressed sample; d) computationally analyzing of the acquired images or videos to calculate the elastic and viscous modulus of the analyzed sample.

[0011] An additional aspect of the invention is a kit comprising:

[0012] • a multi-well plate;

[0013] • at least one glass slide of known and predetermined weight, where the diameter of the glass slide is smaller than that of the well and where the exact center of the glass slide can be indicated with a graphic symbol; • an image acquisition device.

[0014] Another aspect of the invention is a device capable of implementing and automating the method of the invention. This device comprises:

[0015] • a camera for image acquisition, connected to a lens, selectable via a lens mount;

[0016] • a motorized transparent stage positioned above the camera, capable of moving mechanically in transverse or longitudinal directions;

[0017] • a transparent container fixed on the motorized stage;

[0018] • a multi-well plate to be inserted into the container, which, through the movement of the motorized stage, allows the positioning of each well within the field of view of the camera lens;

[0019] • a lid that closes the plate;

[0020] • at least one cylindrical guide inserted into the multi-well plate;

[0021] • mechanical or spring-loaded release mechanisms attached to the lid of the multi-well plate or to the cylindrical guide, which house glass slides of predetermined weight and allow the transfer of the slides into the well to compress the sample;

[0022] • a processor capable of analyzing the images acquired by the camera to calculate the elastic and viscous modulus.

[0023] DESCRIPTION OF THE FIGURES

[0024] Figure 1 illustrates: (A) A schematic of a cellular spheroid, showing the zones of necrosis, quiescence, and proliferation (from the inside out), due to the presence of chemical gradients (nutrients and catabolites); (B) The schematic of the compression test of the same, performed with microscope slides in a cell culture plate.

[0025] Figure 2 illustrates: (A) The processing of transient images of a loaded spheroid for reconstructing the local deformation profile and verifying the correct application of the load; (B) An image of the same spheroid without and with load (top and bottom in the figure, respectively); (C) The optical density profile (I / Io) within a cellular spheroid plotted against its radius (indicated as R) as a function of the applied load (o, specified in the legend) for determining the thickness of the spheroid and calculating the deformation.

[0026] Figure 3 shows the transient deformation of a cellular spheroid following the application of a microscope slide used as a load (creep test) and the transient response following the removal of the load (creep recovery test).

[0027] Figure 4 presents the diagram of the applied compression stress (o) to a cellular spheroid as a function of its deformation (E), measured under steady-state conditions through analysis of images acquired by microscopy, some of which are provided for illustrative purposes. Figure 5 illustrates the data for calculating mechanical properties of spheroids (outlined in the table on the right, (C)) of the same cell line using the Burgers model (A) and the foam model (B).

[0028] Figure 6 illustrates a schematic of the implementation of the invention for performing the rheo-optical compression test in a multi-well cell culture plate.

[0029] Figure 7 shows a technical drawing of the cylindrical guide.

[0030] Figure 8 shows the cylindrical guides, built with a 3D printer, and a multi-well plate with 12 wells containing them.

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] The present inventors have surprisingly discovered a rheo-optical method for mechanical characterization through compression based on inexpensive and accessible materials and equipment, which does not require specific technical expertise for its implementation. It has been found that mechanical characterization of cellular spheroids and biopsies of biological tissues can be carried out by:

[0033] • using samples without special preparations;

[0034] • conducting compression tests with a load (weight) inside standard cell culture plates;

[0035] • applying the load (weight) using economical and readily available equipment;

[0036] • conducting the tests in a sterile environment;

[0037] • acquiring images of the sample and analyzing them to calculate the elastic and viscous modulus.

[0038] In the present invention, load and weight are used interchangeably.

[0039] The subject of the invention is a rheo-optical method for calculating the elastic and viscous modulus of cellular spheroids and biopsies, which comprises the following steps: a) inserting a sample to be analyzed into a well of a cell culture plate; b) applying a compressive force to the sample; c) acquiring single images or videos of the compressed sample; d) computationally analyzing the acquired images or videos to calculate the elastic and viscous modulus of the analyzed sample.

[0040] In the present document, rheo-optical method refers to a method that allows the study of the deformation characteristics, under the action of external forces, of solid, fluid, and semifluid bodies with reference to their properties and conditions (density, viscosity, concentration, temperature, applied forces, etc.) with the acquisition of images through optical elements (microscopes, cameras, video cameras, etc.). The method devised allows for the combination of various useful elements, such as: the imposition of compressive force through the application of a common microscope coverslip as the load (weight), image analysis to calculate the thickness of the cellular spheroid and to determine maps of local deformation, fitting of data with rheological models to extract the mechanical parameters of interest.

[0041] In this application, 'compressive force' refers to the compression associated with the load applied along the longitudinal axis of the system under examination.

[0042] In one embodiment, at step a), the plate is a single- or multi-well plate. In another embodiment, the samples are present along with a suspension medium, a culture medium, or a buffer. A suitable cell culture medium may be, for example, Dulbecco’s Modified Eagle’s Medium (DMEM) enriched with 10% (v / v) Fetal Bovine Serum (FBS), 1% (v / v) antibiotics (50 units / mL penicillin and 50 mg / mL streptomycin), and 1% (v / v) L-glutamine.

[0043] In an another embodiment, at step b), the compressive force is a weight inserted into the well of the sample under examination. The weight is added so that the center of gravity is centered with respect to the sample to be analyzed. The application of an appropriate and centered load (weight) allows the sample to take on a cylindrical shape, which is more conducive to subsequent computational analysis for the determination of mechanical properties. In a preferred aspect, the weight is provided by at least one coverslip or slide. Coverslips or slides typically have a square size of 24 mm x 24 mm with a thickness of 0.130 mm and a weight around 0.2g. The coverslips can be manually placed between the cylindrical guides proposed in the present invention, so as to facilitate their centering with respect to the spheroid, during their sedimentation by gravity. Alternatively, a series of coverslips (from 1 to 10, for example) can be stacked in a release mechanism integral with the cylindrical guide, or connected to the plate cover, which subsequently releases the coverslips individually through an electromechanically controlled automatic mechanism.

[0044] One of the advantages of the present invention is that the method is performed directly within laboratory plates. The relatively short duration of the method prevents the cellular spheroid from adhering to the surface of the plate. However, the bottom of the plate can also be coated with a thin layer of a material that prevents cellular adhesion, such as agarose gel, following common laboratory procedures.

[0045] Another advantage of the present invention lies in the integration of a specially designed guide to conform to the dimensions and shape of commonly available commercial plates. This solution indeed facilitates the application of loads, using only easily accessible and inexpensive materials, such as microscope coverslips, while achieving increased functionality and efficiency. Additionally, the integration of a guide allows for precise management of load application through the microscope coverslips. In particular, the guide ensures proper alignment of the coverslips, thus allowing for uniform load application. This prevents asymmetric stacking of microscope coverslips, thereby improving load distribution. In fact, a significant technical problem affecting the prior art occurs when the coverslip descends unevenly onto the spheroid, resulting in load application in directions other than the orthogonal direction and applied only to a specific portion of the spheroid. This uneven load distribution leads to the development of an asymmetric deformation map, thus distorting the correlation between load and deformation and producing inaccurate quantification of the elastic modulus.

[0046] In another embodiment, at step c), images and / or videos are acquired using a microscope connected to a camera or using a smartphone with an integrated camera; the images and / or videos are also acquired directly or with the assistance of additional lenses. In a preferred aspect, video acquisition is in time-lapse mode.

[0047] Specifically, the use of additional lenses allows for a more precise quantification of the data from the acquired images and / or videos, leading to a better characterization of the shapes of the spheroids after the application of the load through the study of their respective edges.

[0048] Furthermore, the time-lapse mode allows for the study of the transient evolution of the morphological response of biological systems, enabling the identification of the time required to reach a condition of steady deformation, and to determine the accurate value of stress-strain correlation, all in a single analysis procedure. A microscope can acquire a high number of images per second. However, since the steady-state condition is generally reached within a few minutes, even a smartphone possesses image acquisition speeds (frame rates) compatible with the method of the present invention.

[0049] In a further embodiment, the images acquired at step c) are analyzed at step d) through image processing, which includes the following steps: di) determining the contact area between the coverslip and the sample, and determining the thickness of the sample after the application of the weight; d2) determining the deformation of the sample, using the contact area and the sample thickness calculated at step di); da) determining the compressive force, calculated by dividing the applied weight by the contact area calculated at step di); d4) using the compressive force calculated at step da) and the deformation of the sample calculated at step d2) to calculate the elastic and viscous modulus through the application of a rheological model based on viscoelastic analysis; preferably the Burgers model or the foam model.

[0050] Other rheological models may include the Maxwell model, the Kelvin-Voigt model, linear models such as Hooke's model, or other models available in the relevant scientific literature.

[0051] The contact area and thickness can be used to calculate the deformation of the sample (s = (dc— d0 / dc), where e is the deformation, de is the diameter with the load and do is the diameter without load, both in transient and steady-state conditions.

[0052] In this context, 'transient' refers to the time evolution of the morphological response (e.g., in terms of deformation) of the system under examination, associated with dissipative phenomena, while 'steady-state' refers to a condition of deformation under the presence of an imposed load that (after the transient phase) no longer varies over time.

[0053] The experiment conducted in transient conditions can be considered as a creep test, where a constant force is applied, and the deformation over time is measured until reaching a steady-state condition. The compressive force data and the sample deformation data are used to calculate the elastic and viscous moduli through the application of a rheological model, such as the Burgers model, which is represented by two combinations of spring and damper elements in series / parallel with each other.

[0054] The elastic modulus is an indicator of the elastic response of the system under examination, approximated as a solid from a mechanical standpoint, while the viscous modulus is an indicator of the viscous response, assuming that the system from a mechanical standpoint can be approximated as a liquid.

[0055] The method described here is suitable for application in a sterile environment, such as a microscopy station equipped with an incubation system for controlling environmental conditions (z.e., temperature and CO2), or in a clinical setting, such as an operating room.

[0056] The invention also encompasses a kit comprising:

[0057] • a multi-well plate;

[0058] • at least one coverslip of known and predetermined weight, where the diameter of the coverslip is smaller than that of the well, and where the exact center of the coverslip can be indicated with a graphical symbol;

[0059] • a device for image acquisition.

[0060] In a typical configuration, for example using a 12-well multiwell plate, with an internal diameter of each well approximately 21 mm, each coverslip in the kit may have, for example, a circular shape with a diameter of approximately 16 mm and a weight of approximately 0.20 g for coverslips with a thickness of 0.125 mm, or approximately 1 g in case of slide coverslips with a thickness of 1 mm.

[0061] In this way, it is possible to simultaneously handle 12 separate samples, allowing for parallel characterization of them. This scalability feature greatly increases efficiency and productivity compared to the known technique. Additionally, regarding the sizes of the coverslips and the multi-well and multi-well plate, the present invention allows for resolving any discrepancies between the sizes of the circular coverslips and the wells by adjusting the dimensions of the guide, particularly the thickness of its side walls. This capability enables precise application of stresses, regardless of dimensional discrepancies between coverslips and wells.

[0062] Another object of the invention is a device capable of implementing and automating the method of the invention. This device includes:

[0063] • a camera for image acquisition, connected to a lens, selectable from a lens mount ring;

[0064] • a motorized transparent table positioned above the camera, which can mechanically move in transverse or longitudinal directions;

[0065] • a transparent container fixed on the motorized table;

[0066] • a multi-well plate to be inserted into the container, which, through the movement of the motorized table, allows the positioning of each well in the field of view of the camera lens;

[0067] • a cover that closes the plate;

[0068] • at least one cylindrical guide inserted into the multi-well plate;

[0069] • mechanical or spring-release means integral with the cover of the multi-well plate or with the cylindrical guide, where coverslips of predetermined weight are housed, allowing the transfer of the coverslips into the well to compress the sample;

[0070] • a computer capable of analyzing the images acquired with the camera for the calculation of the elastic and viscous modulus.

[0071] EXAMPLES

[0072] In this section, we will refer essentially equivalently to samples of cellular spheroids and biopsies. The spheroid exhibits a more or less heterogeneous structure depending on its dimensions (Figure 1A). An inner core predominantly consists of a necrotic zone where cell survival is hindered by the low presence of nutrients and the difficulty of removing metabolic waste due to the slow transport processes, which possess an essentially diffusive nature. Surrounding the core is a transition zone that then transforms into the outermost layer where cellular proliferation is more active due to the availability of nutrients and the ease of waste removal. Figure IB schematically illustrates the procedure for applying the load, which involves inserting a microscope coverslip into the well where the cellular spheroid to be analyzed was previously placed. The coverslips can be manually placed between the cylindrical guides proposed in the present invention, facilitating their centering with respect to the spheroid during gravity sedimentation. Alternatively, a series of coverslips (from 1 to 10, for example) can be stacked and then released individually through an electromechanically controlled automatic mechanism.

[0073] To simplify the correct application of the load, it's possible to draw or screen-print an "X" or another symbol at the center of the coverslip. Using a stereo microscope or a smartphone to magnify the image ensures that the spheroid is positioned at the symbol drawn or screen- printed on the coverslip, thus ensuring centered loading. Any centering errors can be identified by observing the sample under the microscope or by verifying after load application using image analysis techniques as illustrated below. As shown in Figure 2A, the images of a creep transient are combined into a single composite image where each pixel represents the highest grayscale level or the highest local contrast in the sequence of images. This creates a point-wise deformation map in the focal plane, for example, at the midpoint along the thickness of the spheroid. Any asymmetries in the deformation profile can be attributed to imperfect load centering or asymmetry in the heterogeneous structure of the spheroid.

[0074] The adjustment of the load is useful to ensure that the compressed spheroid takes on a cylindrical shape, which is advantageous for subsequent analyses. Adjustment is particularly necessary when the sample is a spheroid with significant deviations from a spherical shape, as illustrated in the images in Figure 2B, or an irregularly shaped biopsy. Once the sample has assumed a cylindrical shape, the change in thickness following the application or removal of the load is simply derived as the ratio of volume to area, if the volume remains constant as the sample is compressed. As discussed in the state of the art section, at a certain load value, a cellular spheroid begins to release water and other components into the surrounding medium, and the assumption of constant volume is no longer valid. For this reason, direct measurement of thickness is performed from the spheroid images, as illustrated in Figure 2C. Since the spheroid is typically darkener than the surrounding medium, an absorbance value is obtained as the ratio between the optical density of the spheroid I and the optical density of the surrounding medium Io, measured using standard image analysis routines. Absorbance is related to thickness by the Lambert-Beer law: where K is an absorbance coefficient and d is the thickness of the sample. To use the Lambert-Beer law, it is necessary to determine the value of K through calibration, which is performed by measuring the thickness of the sample by focusing on the surfaces of the coverslip and the plate in an area outside the cellular spheroid. The difference in elevation between the two surfaces multiplied by the refractive index of the suspending medium provides the value of the thickness of the cellular spheroid. Given I, Io e d, the value of ean be calculated from the Lambert-Beer equation and used as long as the properties of the spheroid do not change, for example, due to water leakage under load.

[0075] The data from a compression test of a cellular spheroid are presented in Figure 3, where the deformation £ = (d0— d) / d0, calculated as the difference between the current thickness d and the initial thickness do of the spheroid divided by the initial thickness, is plotted against time both in the loading phase (creep) and the recovery phase (creep recovery). The applied compression force (o) is calculated as the ratio between the weight of the coverslip (FP) minus the buoyancy force (FA) and the contact area between the spheroid and the coverslip surface determined from image analysis (cr = FP— FA / A). The steady-state values of compression stress (cr) and strain (E) in a series of tests on different cellular spheroids are shown in Figure 4, where the error bars represent the standard deviation of the results. The same figure also includes some representative images of the steady-state shape of a spheroid at different compression stress values. The images demonstrate a gradual flattening of the spheroid under increasing loads.

[0076] In Figure 5, the analysis of the experimental data from the previous figure is illustrated using the Burgers model (Figure 5A), the foam model (Figure 5B), and the data comparison (Figure 5C). The combinations of elastic elements and dampers underlying the two models are schematically represented in their respective diagrams. The Burgers model applied to the linear region of the stress (o) vs. strain (E) data provides a value of the elastic modulus Eo of 1.5x103Pa, within the range of values available in the literature. The foam model describes well the stress (o) vs. strain (s) data across the entire explored deformation range with the values of the four parameters reported in Figure 5.

[0077] A device capable of implementing the invention is illustrated in Figure 6, where a system for performing the rheo-optical method in a multi-well plate for cell cultures is presented. The system consists of a container on which the plate is placed and is moved by a motorized transparent table for positioning each well in the camera's field of view (either of a smartphone or other). The camera is connected to a lens, possibly selectable from a lens mount ring, to achieve the required magnification. Image acquisition can be performed in bright field or fluorescence mode using suitable illumination and optical components. The plate is closed by a cover and has cylindrical guides where the microscope coverslips are housed. A mechanical release mechanism allows transferring the coverslips into the well to compress the sample uniformly. For example, by using an electromechanically controlled automatic mechanism, a series of coverslips (from 1 to 10, for instance) can be stacked and then released individually. The images acquired by the camera are analyzed and processed as illustrated previously to determine the mechanical properties of each sample. In this way, image acquisition is performed perpendicular to the direction of the stress.

[0078] This configuration offers different and improved evaluations and measurements compared to conventional methods.

[0079] Figures 7 and 8, on the other hand, depict the cylindrical guides. Specifically, Figure 7 displays a technical drawing of a cylindrical guide according to the invention, while Figure 8 depicts a prototype printed with a 3D printer and inserted into a 12-well multi -well plate, as an example.

Claims

CLAIMS1. Rheo-optical method for calculating the elastic modulus and the viscous modulus of cellular spheroids and biopsies, comprising the following steps: a) inserting a sample to be analyzed into a well of a cell culture plate; b) applying a compression force to the sample; c) acquiring single images or videos of the compressed sample; d) computationally analyzing the acquired images or videos to calculate the elastic modulus and the viscous modulus of the analyzed sample.

2. A method according to claim 1, wherein at step a) the plate is a single or multi -well plate and wherein the samples are present together with a suspension medium, a culture medium, or a buffer.

3. A method according to claims 1 to 2, wherein the compression force in step b) is a weight whose center of gravity is centered with respect to the sample, preferably the weight is provided by at least one cover slip or slide.

4. A method according to claims 1 to 3, wherein the acquisition of images or videos at step c) is performed by a microscope connected to a camera or by a smartphone with an integrated camera.

5. A method according to claim 4, wherein the acquisition of images and / or videos is performed directly or through the use of additional lenses, preferably the acquisition of videos is in time-lapse mode.

6. A method according to claims 1 to 5, wherein the acquired images and / or videos in step c) are analyzed in step d) by image processing comprising the following steps: di) determining the contact area between the cover slip and the sample, and determining the thickness of the sample after the application of weight; d2) determining the deformation of the sample, using the contact area and the sample thickness calculated in step di); da) determining the compression force, calculated by dividing the applied weight by the contact area calculated at step di); d4) using the compression force calculated at step da) and the sample deformation calculated at step d2) to calculate the elastic and viscous modulus through the application of a rheological model based on viscoelastic analysis; preferably the Burgers model or the foam model.

7. A method according to claim 4, comprising the movement in transverse or longitudinal direction of a motorized transparent table positioned above the camera, on which said well of a cell culture plate is positioned, for positioning said well in the field of view of said camera.

8. A method according to claim 1, wherein the compression force at step b) is a weight provided by at least one cover slip or slide, said step b) of applying a compression force to the sample comprising the step of placing said cover slips or slides between a cylindrical guide, to facilitate the centering of said cover slips or slides with respect to a cellular spheroid.

9. A method according to claims 1 or 8, comprising the step of stacking a series of said cover slips or slides, of predetermined weight, in a release mechanism; said release mechanism comprising mechanical release means, or spring release means, connected to a cover of said multi-well plate, or to said cylindrical guide, where said cover slips or slides, of predetermined weight, are housed, to allow the transfer of said cover slips or slides into said well, in order to compress said sample.

10. A kit compri sing :• a multi-well plate;• at least one cover slip or slide of known and predetermined weight, where the diameter of the cover slip or slide is smaller than that of the well and where the exact center of the cover slip or slide can be indicated with a graphical symbol;• a device for image acquisition.

11. A kit according to claim 10, wherein the cover slip or slide is circular in shape, has a diameter of 16 mm, and weighs 0.20 g in the case of cover slips of thickness 0.125 mm; or approximately 1 g in the case of slides of thickness 1 mm.

12. A device capable of implementing and automating the method according to claims1 to 6; said device comprising:• a camera, for image acquisition, connected to a lens, selectable from a lens mount;• a motorized transparent table, positioned above the camera, which is able to move mechanically in a transverse or longitudinal direction;• a transparent container fixed on the motorized table;• a multi -well plate to be inserted into the container which, through the movement of the motorized table, allows the positioning of each well within the field of view of the camera lens;• a cover which closes the plate;• at least one cylindrical guide, inserted into the multi-well plate;• mechanical or spring release means, connected to the lid of the multi-well plate, or to the cylindrical guide, where cover slips or slides of predetermined weight are housed, which allow the transfer of the cover slips or slides into the well, in order to compress the sample.• a computer capable of analyzing the images.

13. A device capable of implementing and automating a rheo-optical method for calculating the elastic and viscous moduli of cellular spheroids and biopsies, said device comprising:• a camera, for image acquisition, connected to a lens, selectable from a lens mount;• a motorized transparent table, positioned above the camera, which is able to move mechanically in a transverse or longitudinal direction;• a transparent container fixed on the motorized table;• a multi -well plate to be inserted into the container which, through the movement of the motorized table, allows the positioning of each well within the field of view of the camera lens;• a cover, which closes the plate;• at least one cylindrical guide, inserted into the multi-well plate;• mechanical or spring release means, connected to the cover of the multi-well plate, or to the cylindrical guide, where cover slips or slides of predetermined weight are housed, which allow the transfer of the cover slips or slides into the well, in order to compress the sample;• a computer capable of analyzing the images.

Images