Method for characterizing the deformability of cells or parts of cells in a cell sample - Patent Application 20070122997

Abstract

The present invention relates to a method for characterizing the deformability of cells or parts of cells in a cell sample, the method comprising the steps of: culturing cells in a microstructured plate having microgrooves with a predetermined width and depth for at least partially engaging the nuclei of the cells in one or more microgrooves, wherein at least one part of the surface of the microgroove is a cell adhesion surface; measuring the fluorescent signals of the nuclei, which have been pre-treated to emit fluorescent radiation; determining a fluorescent intensity profile and at least one morphological parameter of the nuclei based on the fluorescent signal measured for each nucleus; and determining a deformation class of the nuclei at the depth of the microgroove based on the fluorescent intensity profile and the at least one morphological parameter.

Description

[Technical Field]

[0001] The present invention relates to a method for characterizing the deformability of cells or parts of cells, in particular cell nuclei, in a cell sample. The present invention also relates to a method for diagnosing a disease state in an individual and to a method for screening candidate compounds for the treatment and / or prevention of a disease state. [Background technology]

[0002] Generally speaking, the mechanical properties of cell nuclei are now considered important biomarkers for many diseases. Currently, the most commonly used laboratory experimental systems for testing these mechanical properties, such as atomic force microscopy, micropipette aspiration, or microflowmeters, are low-throughput, technically complex, and / or costly in terms of both equipment and time. Novel microfluidic experimental systems using the circulation of cells in channels have been developed. They allow for higher throughput, but remain complex.

[0003] From the paper Antmen E et al.: Amplification of nuclear deformation of breast cancer cells by seeding on micropatterned surfaces to better distinguish their malignancies. Colloids Surf B Biointerfaces. 2019 Nov 1;183:110402. doi:10.1016 / j.colsurfb.2019.110402. Epub 2019 Jul 30. PMID:31398621, it is known to deposit cancer cells whose nuclei are fluorescent on a plate with protruding microreliefs in the form of parallel pillars of a size smaller than the cells, and to determine, by analyzing the fluorescence images of the plate, certain morphological parameters of the nuclei related to the deformation of the nuclei in the plate by the pillars, in order to deduce from the same whether the cells are metastatic or healthy. Such fluorescence image analysis is complicated by the variety of shapes that cell nuclei can assume on such plates.

[0004] From the paper Alvarez-Elizondo MB et al., Micropatterned topographies reveal measurable differences between cancer and benign cells. Med Eng Phys. 2020 Jan;75:5-12. doi:10.1016 / j.medengphy.2019.11.004. Epub 2019 Nov 25. PMID:31780301, it is known to deposit cancer cells with fluorescent nuclei in plates with parallel microgrooves smaller than the cells and observe their morphology in the plane of the plate and the orientation of the cells and their nuclei in the direction of the microgrooves to infer from them whether the cells are metastatic or healthy. Such observations are made by adjusting the contour ellipse for each nucleus and determining the major and minor axes of each nucleus, their area, their eccentricity, and their orientation in the microgrooves for a very specific plate topography that must be precisely determined in advance. However, information on deformation in depth has not been investigated. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Antmen E et al.: Amplification of nuclear deformation of breast cancer cells by seeding on micropatterned surfaces to better distinguish their malignancies. Colloids Surf B Biointerfaces. 2019 Nov 1;183:110402. doi:10.1016 / j.colsurfb.2019.110402. Epub 2019 Jul 30. PMID:31398621 [Non-patent document 2] Alvarez-Elizondo MB et al. Micropatterned topographies reveal measurable differences between cancer and benign cells. Med Eng Phys. 2020 Jan;75:5-12. doi:10.1016 / j.medengphy.2019.11.004. Epub 2019 Nov 25. PMID:31780301 Summary of the Invention [Problem to be solved by the invention]

[0006] There is a need for a simple, powerful, high-throughput method for characterizing the mechanical deformation properties of cell nuclei that is reliable, easy to implement in clinical practice, and relatively inexpensive, in order to enable rapid and reliable functional tests to be performed, in particular to enable detection of diseased or non-disease states in cell samples, for example for the purpose of making a diagnosis or screening compounds of interest. [Means for solving the problem]

[0007] The present invention addresses this need by providing a method for characterizing the deformability of cells or parts of cells in a cell sample, wherein said cells each comprise a body and a nucleus: - culturing the cells in a microstructured plate having a plurality of microgrooves on its surface, the microgrooves having a predetermined width, spacing and depth to allow at least partial engagement of a nucleus of at least one of the cells in one or more of the microgrooves, at least a portion of the surface of the microgroove being an adhesive surface for the cells; - measuring the fluorescent signal of the nuclei of said cells by microscopy, the nuclei of said cells having been previously treated to emit fluorescent radiation, - determining a fluorescence intensity profile for each nucleus along at least one axis of said nucleus and at least one morphological parameter of said nucleus based on the fluorescence signal measured for each nucleus; - determining a deformation class of each nucleus at the depth of one or more microgrooves based on the fluorescence intensity profile and at least one morphological parameter determined for said nucleus; The method includes:

[0008] As previously mentioned, the nuclei of cells can be deformed on the adhesive surface of the microstructure. The deformation of the nuclei is determined by the mechanical and biological properties of the cells, such as their stiffness or their contractility. Such deformation characteristics can vary depending on the physiological or disease state of the cell type. Therefore, by studying the deformation characteristics of the cell nuclei from a sample, it is possible to determine the biological state of the cell sample, especially the disease state.

[0009] The presence of microgrooves in a microstructured plate with an adhesive surface allows for the generation of specific deformation stresses on cells, controlled by the dimensions of the microgrooves on the plate. Indeed, cell adhesion to the walls of the microgrooves leads to the deformation of the nucleus with several known morphologies and / or configurations, and in some cases even to the complete entrapment of the nucleus at the depth of the microgrooves. This allows for the determination of various deformation classes of nuclei. By determining the deformation class for each nucleus, the percentage of cells classified into a deformation class compared to a reference sample can be compared to infer biological properties therefrom.

[0010] A comparative study of statistical intensity or morphometric variables, in particular the distribution of parameters of fluorescence intensity profiles or the distribution of morphological parameters, of the nuclei of cells in a sample classified into at least one variation class makes it possible to determine the biological properties of the sample with greater confidence than a comparative study of said statistical intensity or morphometric variables for all the nuclei of cells in the sample.

[0011] The method may be an ex vivo or instant method.

[0012] The method is non-therapeutic in nature.

[0013] Microstructure Plate The microstructured plate preferably comprises a transparent support, in particular made of glass, and a microstructured polymer structure, in particular made of polydimethylsiloxane (PDMS), with microgrooves on its surface.

[0014] The microgrooves are preferably parallel to one another.

[0015] The microgrooves preferably have a predetermined width and depth so as to cause deformation of a portion of the cell in the direction of the depth of the microgrooves.

[0016] The width, depth and spacing of the microgrooves may be selected based on at least one physical parameter of at least some cells of a cell type, particularly the dimensions of the cells, the dimensions of the nuclei of the cells, and / or biophysical properties of the cells, particularly the stiffness, adhesive strength or contractility of the cells. The width, depth and spacing of the microgrooves may be selected to have an average percentage of nuclei fully captured in the microgrooves based on a given cell type in a sample.

[0017] The average percentage of nuclei of healthy or abnormal cells of said cell type that are completely trapped in the microgrooves may be between 5% and 95%, and even better between 10% and 90%.

[0018] The width, depth and spacing of the microgrooves are preferably 30 to 60% of the average minor axis length of the nuclei of non-transformed cells.

[0019] "Average minor axis length" means the average length of the minor axis of the ellipsoid adjusted to the shape of the nucleus of non-transformed cells.

[0020] The width of the microgrooves is predetermined for the cell type and varies based on the biological properties of the cell type being studied. Generally speaking, the width is greater than or equal to 3 μm and / or less than or equal to 10 μm.

[0021] The depth of the microgrooves is predetermined for each cell type and varies based on the biological characteristics of the cell type being studied. Generally speaking, the depth is greater than or equal to 4 μm and / or less than or equal to 10 μm. For muscle cells, the depth may be 4-5 μm to achieve a sufficiently large nuclear content in the microgrooves. For breast epithelial cells, the depth may be 7-9 μm, e.g., approximately equal to 8 μm. All microgrooves may have a substantially constant depth along their entire length.

[0022] The microgrooves may have spacing between them of 3 μm or more and / or 10 μm or less, measured between adjacent edges.

[0023] All of the microgrooves may have a substantially constant width over their entire length. All of the microgrooves may have a substantially constant depth over their entire length.

[0024] All of the microgrooves may be approximately uniformly spaced along their entire length.

[0025] Alternatively, the microstructured plate may include distinct compartments, each containing microgrooves of varying width and / or spacing.

[0026] The depth may be fixed or may vary on the microstructured plate and / or along the microgrooves.

[0027] At least a portion of the inner surface of the microgrooves, in particular the sidewalls and / or bottom of the microgrooves, preferably the entire surface of the microstructured plate, may be coated with an adhesive coating, in particular a cell adhesion protein, such as fibronectin, collagen, laminin or gelatin.

[0028] Cell samples The cells may be adherent cells, such as muscle cells, endothelial cells, stem cells, preferably non-embryonic or non-human stem cells, epithelial cells, nerve cells, bone cells, adipocytes, podocytes, cancer cells or a mixture of such cells.

[0029] The method may include a step of fixing the cells to the microstructure plate by adding an alcohol compound, particularly methanol, or an aldehyde compound, particularly paraformaldehyde. The step of fixing the cells may be performed at least one hour after the sample is deposited on the microstructure plate. Such a length of time allows the cells time to deform before being fixed, with the result being indicative of the deformation of the cells in the sample.

[0030] The method may comprise the step of treating the cell sample to cause the nuclei to emit the above-mentioned fluorescent radiation, which may be carried out before or after the cells of the sample are placed on the microstructure plate, preferably after the cells of the sample are placed on the microstructure plate, and even better after the cells are fixed on the microstructure plate.

[0031] The nuclei of the cells of the sample may be labeled using a fluorescent nuclear label, in particular Hoechst stain or DAPI, before or after fixation.

[0032] In a variant, treating the cells to cause the nucleus to emit fluorescent radiation may involve transducing the cells with a plasmid encoding a nuclear protein fused to a fluorescent protein.

[0033] The cell sample may be of human, animal or plant origin.

[0034] The cell sample is preferably constructed so that the cell density of the cell sample during microscopy measurement avoids cell confluence, in particular so that the cell confluence is 60% or less. For example, for muscle, endothelial or epithelial cells, the cell density of the sample deposited on the microstructure plate is 10,000 cells / cm. 2 and / or 50,000 cells / cm 2 It may be the following:

[0035] Microscopy measurements The microscopy measurement may involve the acquisition of a fluorescence image of the surface of the microstructured plate. The fluorescence image obtained may be an image from above or below due to the transparency of the microstructured plate, and may in particular be obtained by epifluorescence.

[0036] In a variant or combination, the microscopy measurements may comprise acquiring fluorescence images in a single plane and / or in multiple planes across the microchannel, in particular by confocal microscopy.

[0037] The microscopy measurement may involve the detection of a fluorescent signal emitted by the nucleus of each cell in the acquired fluorescent image.

[0038] Image analysis The method, in particular the step of determining the fluorescence intensity profile and the morphological parameters, may comprise the step of detecting nuclei in the image and also the contours of said nuclei, in particular based on the fluorescence signals emitted by the nuclei visible in the acquired fluorescence image and on a morphometric analysis of said shape of the nuclei.

[0039] The steps of determining the fluorescence intensity profile and at least one morphological parameter, and determining the deformation class may all be performed automatically, in particular by a processor running software for processing the fluorescence images obtained by microscopy measurements. The processor may include automatic or deep learning software.

[0040] Determination of fluorescence intensity profile A fluorescence intensity profile may be determined based on the fluorescence signal, in particular on the acquired fluorescence image, perpendicular to the axis of extension of the microgrooves, preferably approximately at the mid-plane of said nucleus.

[0041] Determination of morphological parameters The at least one morphological parameter of the nucleus of the cell may be selected from the circularity, roundness, solidity, aspect ratio, and / or ratio of elliptical Fourier coefficients of the nucleus, and / or negative mean curvature of the nuclear contour.

[0042] "Negative mean curvature" means the mean concave curvature of the nuclear contour.

[0043] Morphological parameters may be selected, particularly by pre-analysis on a reference sample, to provide one or more relevant parameters for distinguishing between various biological characteristics of the sample, particularly between diseased and healthy characteristics. Morphological parameters are selected such that for a plurality of morphological parameters or the distribution of each morphological parameter, a statistical difference is established between samples with various biological characteristics, particularly healthy and diseased samples.

[0044] Determining the transformation class The method preferably comprises determining, based on the fluorescence intensity profile and at least one morphological parameter for each nucleus: a) freely suspended nuclei corresponding to one or more morphological parameters characteristic of a less deformed and / or less tortuous nuclear contour, such as a high degree of circularity, roundness, solidity and / or ratio of elliptical Fourier coefficients, in particular above a respective predetermined threshold, and / or a small aspect ratio and / or negative mean curvature, in particular below a respective predetermined threshold, e.g. an approximately horizontal fluorescence intensity profile or a profile lacking large fluorescence intensity peaks; b) deformed nuclei, elongated into at least two adjacent microgrooves, corresponding to a fluorescence intensity profile with, for example, at least two main peaks, associated with at least one morphological parameter characterizing a significant distortion of the contour, in particular a small solidity compared to a predetermined threshold value, and / or a small ratio of elliptical Fourier coefficients compared to a predetermined threshold value, and / or a large negative mean curvature compared to a predetermined threshold value, and / or a small circularity and / or roundness compared to a predetermined threshold value, c) captured nuclei, in particular fully inserted into one microgroove, corresponding to a fluorescence intensity profile with, for example, one main peak, associated with at least one morphological parameter characterizing the elongation of the nuclei, in particular a small circularity and / or roundness compared to a predetermined threshold and / or a large aspect ratio compared to a predetermined threshold. determining a deformation class of the nucleus at one or more micro-groove depths from at least three predetermined deformation classes corresponding to the respective deformation classes;

[0045] The method may comprise comparing at least one statistical property for at least one deformation class obtained for nuclei of cells from the sample with the same property in a reference sample, The statistical property may be the proportion of cells in the cell sample that fall into the at least one deformation class and / or a statistical or morphometric variable for the nuclei of cells classified into the at least one deformation class.

[0046] The method may comprise determining the proportion of cells in the cell sample that fall into at least one of the variant classes, in particular the class of trapped and / or variant nuclei. The method may comprise determining a biological property of the cell sample by comparing the proportion of cells in the cell sample that fall into a plurality of variant classes, or each variant class, with the proportion of cells that fall into said at least one variant class in a reference sample of the same cell type, the biological property of the reference sample being known.

[0047] The method may comprise a step of determining a statistical distribution of at least one or a plurality of morphological parameters for nuclei of cells from a sample classified into one of the deformation classes, in particular the class of deformed nuclei and / or the class of trapped nuclei. The method then preferentially comprises a step of comparing a variable of said statistical distribution of the sample with the same variable of the statistical distribution of morphological parameters for cells classified into said same deformation class in a reference sample whose biological properties are known. The inventors have shown that by limiting the morphological comparison to cells of a specific deformation class in the sample, it is possible to achieve a more accurate biological characterization of the sample compared to when all cells in the sample are used.

[0048] The biological characteristic may be a diseased or non-diseased characteristic of the cell sample.

[0049] The method may include a prior step of determining a variant class, wherein the proportion of cells classified into said variant class, measured relative to a reference sample, allows the best possible distinction between at least two different populations with respect to a biological property.

[0050] Alternatively, or in addition, the method may include a prior step of determining the combination of one or more morphological parameters of nuclei and one or more deformation classes that allows the best possible discrimination between two reference populations with different biological properties, based on the variance of the statistical distribution of the morphological parameters for the nuclei classified into the deformation classes.

[0051] This or these pre-determining steps may include one or more statistical tests for comparing the samples.

[0052] Diagnostic methods The present invention provides a method for diagnosing a disease state in an individual, comprising at least the following steps: a) culturing a sample of a cell type isolated from said individual, wherein cells of the sample include somas and nuclei, and wherein the cells are cultured in a microstructured plate having a plurality of microgrooves on its surface, at least a portion of the surface of the microgrooves being an adhesive surface for the cells, the microgrooves being of a predetermined width and depth to allow at least partial engagement of the nucleus of at least one of said cells in one or more of the microgrooves; b) measuring by microscopy the fluorescent signal of the nuclei of cells from the sample, the nuclei of the cells being pre-configured to emit fluorescent radiation; c) determining a fluorescence intensity profile for each nucleus along at least one axis of said nucleus and at least one morphological parameter of said nucleus based on the fluorescence signal measured for each nucleus; d) determining a deformation class of each nucleus at the depth of one or more microgrooves based on the fluorescence intensity profile and at least one morphological parameter determined for said nucleus; e) comparing at least one characteristic of at least one variant class of nuclei of cells from the sample obtained in step d) with the same characteristic of a reference sample in order to reach a conclusion therefrom regarding the disease state of the individual. This need is also met by a method, including:

[0053] The properties described above in relation to the method for characterizing the deformability of a cell or a part of a cell apply to this diagnostic method in combination with each other or independently of each other and independently of the method for characterizing the deformability of a cell or a part of a cell.

[0054] Screening Method The present invention provides a method for screening candidate compounds for the treatment and / or prevention of a disease state, comprising at least the following steps: a) in vitro culturing of a first sample of a cell type representative of a disease in the absence of a candidate compound; b) in vitro culturing of a second sample of said cell type representative of said disease in the presence of a candidate compound; an in vitro culture, wherein cells of a first and second sample include somas and nuclei and are cultured in first and second identical microstructured plates, each having a plurality of microgrooves on its surface, at least a portion of the surface of the microgrooves being an adhesive surface for the cells, the microgrooves being of a predetermined width and depth to allow at least partial engagement of a nucleus of at least one of the cells in one or more of the microgrooves; c) measuring by microscopy the fluorescent signals of the nuclei of the cells from the first and second samples, wherein the nuclei of the cells from the first and second samples are pre-configured to emit fluorescent radiation; d) determining a fluorescence intensity profile along at least one axis of the nucleus and at least one morphological parameter of the nucleus for each cell from the first and second samples based on each measured fluorescence signal; e) determining a deformation class of the nucleus of each cell from the first and second samples in the direction of the depth of the microchannel based on the determined fluorescence intensity profile and the at least one determined morphological parameter; and f) comparing at least one characteristic of the first sample with the same characteristic for the second sample in at least one variant class of nuclei of cells from the first sample, wherein observing a difference between said characteristics of the first and second samples indicates the effectiveness of the candidate compound against said disease. This need is also met by a method, including:

[0055] The method may include culturing a third sample of the cell type believed to be healthy in the absence of the candidate compound; measuring fluorescent signals of nuclei of cells from the third sample by microscopy, wherein the nuclei of the cells of the third sample are pre-configured to emit fluorescent radiation; determining a fluorescent intensity profile along at least one axis of the nucleus and at least one morphological parameter of the nucleus for each cell from the third sample based on each measured fluorescent signal; determining a deformation class of the nucleus of each cell from the third sample in the direction of the depth of the microgroove based on the determined fluorescent intensity profile and the at least one determined morphological parameter; and comparing at least one characteristic of the first sample and / or the second sample in at least one deformation class of nuclei of cells from the first sample and / or the second sample with the same characteristic for the third sample, wherein observing a difference between the characteristics of the first and third samples and / or a similarity between the characteristics of the second and third samples indicates the effectiveness of the candidate compound for the disease.

[0056] The properties described above in relation to the method for characterizing the deformability of a cell or a portion of a cell apply to this screening method in combination with each other or independently of each other and independently of the method for characterizing the deformability of a cell or a portion of a cell.

[0057] Preferably, the characteristic is the proportion of cells in the sample that fall into at least one of the variant classes, in particular the class of captured nuclei, and observation of a statistical difference between the first and second samples, and / or observation of an insignificant statistical difference between the healthy sample and the sample in the presence of the candidate compound, and observation of a significant statistical difference between the healthy sample and the sample without the candidate compound indicates the effectiveness of the candidate compound for said disease.

[0058] The characteristic is a statistical distribution of a plurality of morphological parameters or at least one morphological parameter for cells in the first and second samples that fall into one of the deformation classes, in particular the class of deformed nuclei or preferentially the class of trapped nuclei. The method then preferentially comprises comparing said statistical distribution of the first sample with the statistical distribution profile of the second sample, wherein observing a statistical difference between the first and second samples and / or observing an insignificant statistical difference between the healthy sample and the sample in the presence of the candidate compound and a significant statistical difference between the healthy sample and the sample without the candidate compound indicates the effectiveness of the candidate compound for said disease. [Brief explanation of the drawings]

[0059] [Figure 1] FIG. 1 shows a schematic diagram of the overall process for characterizing the deformability of cells or cell parts in a cell sample. [Figure 2A] FIG. 1 shows a cross-sectional detail of a microstructured plate holding cells, the nuclei of which are trapped in the microgrooves. [Figure 2B] 1 shows a perspective view and a cross-sectional view along AA of a microstructured plate holding cells, the nuclei of which are visible in fluorescence. [Figure 2C] 1. Perspective and cross-sectional views along AA of a microstructured plate holding cells, with the cell bodies visible in fluorescence. [Figure 3] Fluorescence images of nuclei acquired for samples of various cell types. [Figure 4] 4 is a graph showing the percentage of nuclei captured as a function of average nuclear volume for some of the cell types from FIG. 3. [Figure 5] FIG. 10 shows examples of transformation classes and fluorescence intensity profiles corresponding to each transformation class. [Figure 6] Fluorescence images were acquired with nuclei of each deformation class identified by different colors. [Figure 7] Fluorescence images of myoblast nuclei acquired for microgrooves of various widths. [Figure 8] FIG. 8 is a boxplot showing the percentage of nuclei captured as a function of width from FIG. 7. [Figure 9] FIG. 8 is a boxplot showing the percentage of deformed nuclei as a function of width from FIG. 7. [Figure 10] Fluorescence images acquired for plates at various depths. [Figure 11] FIG. 11 is a boxplot showing the percentage of nuclei captured as a function of depth from FIG. 10. [Figure 12] FIG. 11 is a boxplot showing the percentage of deformed nuclei as a function of depth from FIG. 10. [Figure 13] Box plot showing the percentage of captured nuclei for healthy (WT) and diseased (MU Lamin A) myoblast samples. [Figure 14] Box plot showing the percentage of deformed nuclei for healthy (WT) and diseased (MU Lamin A) myoblast samples. [Figure 15] Box plot showing the percentage of captured nuclei for a healthy breast epithelial cell sample (MCF10A) and a diseased breast epithelial cell sample (MCF7). [Figure 16] FIG. 1 shows the statistical distribution of circularity determined for all cells of a healthy myoblast sample (WT) and for cells with trapped nuclei (b) and cells with deformed nuclei (c) for a diseased myoblast sample (MU). [Figure 17]FIG. 1 shows the statistical distribution of the ratios of elliptic Fourier coefficients determined for all cells of a healthy myoblast sample (WT) and a diseased myoblast sample (MU) for cells with trapped nuclei (b) and cells with deformed nuclei (c). DETAILED DESCRIPTION OF THE INVENTION

[0060] FIG. 1 shows the overall steps for characterizing the deformability of cells or parts of cells in a cell sample.

[0061] The cell sample may be a sample of adherent cells comprising muscle cells, endothelial cells, epithelial cells, nerve cells, bone cells, adipocytes, podocytes, cancer cells or a mixture of said cells. The cells may be human, animal or plant cells.

[0062] In step 10, the cell sample is deposited on the surface of a microstructure plate 12, illustrated in Figures 2A-2C, and cultured on the surface of plate 12 in a suitable medium for a period of at least 1 hour.

[0063] Plate 12 comprises a transparent support 14, particularly made of glass, and a microstructured polymer structure 16, particularly made of polydimethylsiloxane (PDMS), fixed to the support. The surface of microstructure 16 has a plurality of microgrooves 18, preferably of constant width l and depth p over their entire length, that are identical, parallel, and uniformly spaced. However, this may be otherwise; the microgrooves may comprise at least two distinct longitudinal portions, each of constant width and depth, differing from one another in terms of depth and / or width, and / or the microstructure plate may comprise at least two distinct sections, each containing microgrooves that are identical within said section but differ between the two sections in terms of microgroove width, depth, and / or spacing.

[0064] The microgrooves 18 have a predetermined width l and depth p that allow at least partial engagement of at least one nucleus 23 of the cells in one or more of the microgrooves. The microgrooves are preferably also dimensioned to prevent the entire body of the cell 22 from engaging the microgroove 18. To this end, the width l and depth p of the microgrooves 18 may be selected based on at least one physical parameter of the cells of the cell type of the sample, in particular the average size of the cells or the average size of their nuclei, and / or biophysical properties of these cells, in particular the average stiffness, average adhesive strength, or average contractility of the cells. The width l and depth p of the microgrooves 18 may be selected such that the percentage of nuclei completely captured in the microgrooves 18 is a parameter indicative of a biological property of the cells of the cell type to be determined, in particular the diseased or non-diseased nature of the cells. For example, in the case of healthy or malignant cells of the cell type, in particular endothelial or muscle cells, the average percentage of nuclei completely captured in the microgrooves may be between 10% and 90%. The width l, spacing e, and depth p of the microgrooves 18 may be 30 to 60% of the average minor axis length of the nuclei of non-transformed cells. The width l of the microgrooves may be 3 to 10 μm, the depth p of the microgrooves may be 4 to 10 μm, and the spacing e between the microgrooves measured between adjacent ends of the microgrooves may be 3 to 10 μm.

[0065] The surface of the microstructure 16 is at least partially treated with a cell adhesive, such as fibronectin 20. Such treatment can be performed by passing plasma through the plate 12 and then incubating it in a solution containing fibronectin. The treatment is preferably performed over the entire surface of the plate 12, although this can be different. It can also be performed only on the bottom and / or sidewalls of the microgrooves 18.

[0066] During the culture of the cells 22, the cells may or may not deform due to the undulations in the plate 12, and the nuclei 23 of the cells fit more or less into the microgrooves of the plate 12.

[0067] The cells 22 are fixed by adding a fixative compound, such as an aldehyde compound, particularly paraformaldehyde. After the cells are permeabilized with a permeabilizing agent such as Triton X-100, the nuclei of the cells are labeled with a fluorescent label such as 4',6-diamidino-2-phenylindole (DAPI). The nuclei of the cells may be labeled differently, particularly by transfecting the cells with a plasmid encoding a nuclear protein fused to a fluorescent protein, or by adding a fluorescent nuclear label such as Hoechst stain before fixation. Fixing the cells allows for overcoming cell movement, as the confinement of the nuclei to the microgrooves can be statistically reversed over time.

[0068] The cell sample is preferably configured such that the cell density of the cell sample when the cells are fixed avoids cell confluence. When the cells are fixed, the cell density is 10,000 to 50,000 cells / cm. 2 may be.

[0069] Then, in step 30, a fluorescent image of the surface of plate 12 is taken using a fluorescent microscope. As shown in Figure 3, the nuclei of the cells and their shapes can be identified by the fluorescence they emit. It can be clearly seen from Figure 3 that this applies to the various cell types mentioned above.

[0070] Analysis, preferably automatic analysis, of the images obtained in step 40 makes it possible to determine, for each identifiable cell nucleus 23, a fluorescence intensity profile along an axis, preferably perpendicular to the long axis X of the microgroove, and approximately in the center of the nucleus.

[0071] This makes it possible to determine a contour for each nucleus 23, in particular by adjusting a shape model, for example an ellipse model, in order to deduce therefrom various parameters of the nucleus shape, in particular the following parameters: - the following formula:

number

number

number

[0072] In step 50, the fluorescence intensity profile and morphological parameters allow each cell whose nucleus 23 can be identified to be classified into a deformation class among a number of previously established deformation classes. In particular, the deformation class may be based on the percentage of penetration of the nucleus into the microgrooves. The classification may be, for example: i) freely suspended nuclei corresponding to one or more morphological parameters characteristic of a less deformed, in particular approximately circular, and / or less twisted nuclear contour, such as a high degree of circularity, roundness, solidity and / or ratio of elliptical Fourier coefficients, in particular close to 1, and / or a small aspect ratio and / or negative mean curvature, in particular below the respective predetermined threshold, for example an approximately horizontal fluorescence intensity profile or a profile lacking large fluorescence intensity peaks; ii) a deformed nucleus, elongated into at least two adjacent microgrooves, corresponding to a fluorescence intensity profile with, for example, at least two main peaks, associated with at least one morphological parameter characterizing a significant distortion of the contour, in particular a small solidity compared to a predetermined threshold, and / or a small ratio of elliptical Fourier coefficients compared to a predetermined threshold, and / or a large negative mean curvature compared to a predetermined threshold, and / or a small circularity and / or roundness compared to a predetermined threshold, iii) captured nuclei, in particular fully inserted into one microgroove, corresponding to a fluorescence intensity profile with, for example, one main peak, associated with at least one morphological parameter characterizing the elongation of the nuclei, in particular a small circularity and / or roundness compared to a predetermined threshold and / or a large aspect ratio compared to a predetermined threshold. It is conducted from three classes corresponding to the above.

[0073] Figure 5 shows example fluorescence intensity profiles for each of the three classes. As can be seen in Figure 5, to aid in classification, it is also possible to perform confocal measurements on each nucleus transversely to the plate and perpendicularly to the grooves, particularly at the mid-plane of the nucleus. Such measurements allow clear visualization of the depth of nuclear penetration, thereby allowing for refinement of classification, particularly during preliminary studies of relevant parameters. However, such measurements are not required.

[0074] As shown in Figure 6, the class of each nucleus may be reported on the fluorescence image using a color code to allow for rapid identification directly on the image.

[0075] There are then several possibilities for determining the biological characteristics of the cell sample, in step 60, particularly their diseased or healthy nature. As can be seen in Figures 13 to 15, it is possible to compare the percentage of cells of the sample that fall into one of these classes, in particular the class of trapped nuclei or deformed nuclei, with the percentage of cells of another reference sample, in particular a healthy sample, whose biological characteristics are known. As can be seen in Figures 16 or 17, it is also possible to compare the statistical distribution of one of the morphological parameters in one of the deformed classes, in particular the class of trapped nuclei or the class of deformed nuclei, with the statistical distribution of a test sample of cells, in particular a healthy sample, whose biological characteristics are known. These various methods may be combined with each other as needed. The method for determining the biological characteristics depends, in particular, on the plate used, the cell type, and the cell characteristics to be determined. The best method for differentiation may be predetermined using statistical tests. These statistical tests are performed under the same general conditions (same plate type, same culture method, and same imaging method), in particular on diseased and healthy samples with biological characteristics known to be different. These preliminary statistical tests are preferentially performed on multiple panels of reference samples to verify the robustness of a particular method.

[0076] Then, depending on the comparison obtained between the sample being tested and the reference sample, in step 70, the biological property is identified as being identical to the biological property of the reference population, or in step 80, the biological property is identified as being different from the biological property of the reference population.

[0077] Such studies have several potential applications. For example, they allow the determination of the diseased or non-diseased nature of a patient-derived sample by comparing it to one or more healthy or diseased reference populations, in order to infer a diagnosis therefrom. Studies can also be used to screen candidate compounds by comparing the results for samples administered the candidate compound with the results of control samples not administered the candidate compound, and optionally by comparing the results of samples administered the candidate compound and control samples not administered the candidate compound with the results of a reference population, particularly a population considered to be healthy. [Example]

[0078] Figures 3 and 4 show studies on various cell types.

[0079] Figure 3 shows the following cell types: a) myoblasts, b) Human umbilical vein endothelial cells (HUVECs) c) COS-7 cells, d) HeLa cells, e) parietal epithelial cells (PEC), f) Mammary epithelial cells (MCF-10A), g) breast cancer cells (MDA-MB-231), h) breast cancer cells (MCF7), and i) podocytes 1 shows images obtained by epifluorescence imaging on top and confocal imaging along section Z on bottom.

[0080] Cells are cultured in microstructured plates with parallel microgrooves 5 μm wide and deep, uniformly spaced 5 μm apart.

[0081] These images show that all the cells studied are deformable in the plate, with the degree of nuclear deformation depending on the cell type.

[0082] Figure 4 shows the mean nuclear volume of the cell, V, for four of the cell types mentioned above for a subset of these cells. m Percentage P of nuclei in a cell sample belonging to the captured nuclei class as a function of (measured in μm in the plane) p Shows.

[0083] Percentage of captured nuclei in the sample, P p is the average nuclear volume V m This explains why there is no simple relationship between nuclear volume and nuclear deformation. [Example]

[0084] Figures 7-12 show a study of the effect of microgroove dimensions on nuclear deformation for myoblast samples.

[0085] In this example, the plate is made of microstructured PDMS on a glass support. The plate is coated with a fibronectin coating over its entire surface. Myoblast cell samples are cultured in the plate in culture medium for 8 h. At the end of the culture period, the cells are fixed with 4% paraformaldehyde for 15 min. After a permeabilization step using Triton, the nuclei are fluorescently labeled with DAPI for 1 h. Images of the nuclei are then taken using a fluorescence microscope (20x lens).

[0086] Figure 7 shows images obtained by epifluorescence imaging of a myoblast sample on a support with microgrooves spaced 5 μm apart, 4 μm deep, and of various widths, the widths being: a) l = 3 μm, b) l = 5 μm, and c) l = 7 μm As stated above.

[0087] 8 and 9 show the percentage of nuclei classified into the captured nuclei classes, P, as a function of the microgroove width, l. p and the percentage of nuclei classified into the deformed nucleus class, P dand , respectively. It is observed that with increasing width of the microgrooves, the proportion of different classes of nuclei changes (a decrease in the percentage of deformed nuclei and an increase in the percentage of trapped nuclei).

[0088] Figure 10 shows images obtained by epifluorescence imaging of a myoblast sample on a support with microgrooves spaced 5 μm apart, 5 μm wide, and of various depths, as follows: a) p = 4 μm, and b) p = 5.4 μm As stated above.

[0089] 11 and 12 show the percentage of nuclei classified into the captured nuclei classes, P, as a function of the microgroove depth, p. p and the percentage of nuclei classified into the deformed nucleus class, P d and , respectively. It is observed that with increasing microgroove depth, the proportion of different classes of nuclei (a decrease in the percentage of deformed nuclei and an increase in the percentage of trapped nuclei) changes.

[0090] Therefore, the dimensions of the microgrooves have a significant effect on the deformation of cells in the microstructured plates, and the dimensions of the microgrooves can be adapted for each cell type based on their deformability, especially through prior studies, to optimize the study of cell deformation. [Example]

[0091] 13 and 14 show the percentage of captured nuclei, P, for a sample of myoblast-type cells from a healthy patient (WT) and a sample of myoblast-type cells from a diseased patient with a mutation in the gene encoding lamin A, a major component of the nuclear envelope (MuLaminADeltaK32). p and the percentage of deformed nuclei P d Cell samples are cultured in plates with microgrooves 5 μm wide and 4 μm deep, spaced 5 μm apart, using the method described in the previous examples.

[0092] Percentage of captured nuclei P p and the percentage of deformed nuclei P d It was observed that the minimum and maximum values ​​of ρ do not intersect between healthy WT and diseased Mu Lamin A samples, indicating significant differences in these parameters between healthy WT and diseased Mu Lamin A samples. This result indicates that the percentage of captured nuclei, P p or the percentage of deformed nuclei P d We show that determining the α-lamin A concentration makes it possible to distinguish between WT and Mu lamin A samples and thus detect disease. [Example]

[0093] FIG. 15 shows the percentage of captured nuclei, P, for a sample of healthy breast epithelial cells (MCF10A) and a sample of cancerous breast epithelial cells (MCF7). p Cell samples are cultured in plates with microgrooves 5 μm wide and 7.5 μm deep, spaced 5 μm apart, for 24 h using the method described in the previous examples.

[0094] The percentage of captured nuclei (P) between the non-cancerous breast epithelial cell MCF10A sample and the cancerous breast epithelial cell MCF7 sample p This result indicates that the percentage of captured nuclei is a good indicator of differentiation even in the case of breast epithelial cells. [Example]

[0095] 16 shows the normalized statistical distribution of circularity for all cells in a sample (a), trapped nuclei only (b), and deformed nuclei only (c) for a sample of myoblast-type cells from a healthy patient (WT) and a sample of myoblast-type cells from a diseased patient with a lamin A mutation (Mu). The cell samples were cultured in plates with microgrooves 5 μm wide and 4 μm deep, spaced 5 μm apart, using the method described in the previous examples.

[0096] The difference d between the maximum values ​​of the two distributions, WT and Mu, was calculated to determine the distribution separation and thereby characterize the ability to distinguish between the two cell samples. The difference d was equal to 0.06 for (a) (whole cells), 0.25 for (b) (trapped nuclei), and 0.23 for (c) (deformed nuclei).

[0097] It is observed that the difference d is much larger in the cases of (b) and (c), confirming that the classification mechanism prior to the nuclear morphological study improves the distinction between the two cell samples WT and Mu.

[0098] Figure 17 shows the same study applied to the elliptic Fourier coefficients. The difference d is equal to 0.12 for (a) (whole cells), 0.31 for (b) (trapped nuclei), and 0.12 for (c) (deformed nuclei).

[0099] A much larger difference d is observed in case (b), confirming that the classification mechanism prior to nuclear morphological study improves the distinction between the two cell samples WT and Mu.

[0100] However, this time better differentiation is possible only in the classes of nuclei captured, confirming that preliminary studies may be useful to determine the appropriate combination of classes and morphological parameters to be studied in order to have the best differentiation of samples with different biological properties.

[0101] The present invention is not limited to the above-described embodiments. For example, the microstructure plate may have microgrooves with a more complex distribution rather than uniform spacing. The plate may include multiple sections with different microgroove characteristics.

[0102] Alternatively, other morphological parameters characterizing the three-dimensional shape of the nucleus can be envisaged, provided that they allow an effective distinction between two cell samples with different biological properties.

[0103] As a variant, the present invention is not limited to the types of cells and diseases mentioned, the methods mentioned have potential for many cell types and many diseases. [Explanation of symbols]

[0104] 10 steps 12 Microstructure Plate 14 Support 16 Microstructured polymer structures 18 Microgrooves 20 Adhesive Coating 22 cells 23 Nuclear 30 steps 40 processes 50 steps 60 processes 70 processes 80 processes

Claims

1. A method for characterizing the deformability of cells (22) or parts of cells (22) in a cell sample, each of said cells (22) comprising a body and a nucleus (23): - culturing the cells (22) in a microstructured plate (12) having a plurality of microgrooves (18) on its surface, the microgrooves (18) having a predetermined width l and depth p that allow at least partial engagement of a nucleus (23) of at least one of the cells (22) in one or more of the microgrooves (18), at least a portion of the surface of the microgrooves (18) being an adhesive surface for the cells; - measuring the fluorescent signal of the nuclei (23) of said cells (22) by microscopy, the nuclei (23) of said cells (22) having been previously treated to emit fluorescent radiation, - determining, based on the fluorescence signal measured for each nucleus (23), a fluorescence intensity profile for each nucleus (23) along at least one axis of said nucleus and at least one morphological parameter of said nucleus (23), - determining the deformation class of each nucleus at the depth of the one or more microgrooves (18) based on the fluorescence intensity profile and at least one morphological parameter determined for said nucleus; A method comprising:

2. 2. The method according to claim 1, wherein at least a portion of the inner surface of the microgrooves (18), in particular the side walls and / or the bottom of the microgrooves, is coated with an adhesive coating (20), in particular a cell adhesion protein, such as fibronectin, collagen, laminin or gelatin.

3. 3. The method according to claim 1 or 2, wherein the cells (22) are selected from adherent cells, such as muscle cells, endothelial cells, epithelial cells, podocytes and / or cancer cells.

4. 4. The method according to any one of claims 1 to 3, wherein the microscopy measurement comprises acquiring a fluorescence image of the surface of the microstructure plate (12).

5. 5. The method according to claim 4, wherein determining the morphological parameters may comprise detecting the contour of the nucleus (23) of the cell (22) in the acquired fluorescence image and performing a morphometric analysis of the shape of the nucleus (23) to deduce the morphological parameters therefrom.

6. 6. The method according to any one of claims 1 to 5, wherein the microscopy measurements comprise acquiring fluorescence images in a single plane and / or in multiple planes across the microchannels (18), in particular by confocal microscopy.

7. 7. The method according to claim 1, wherein the fluorescence intensity profile is determined in the fluorescence signal, in particular in the acquired fluorescence image, perpendicular to the axis of extension of the microgrooves (18), preferably approximately at the central plane of the nucleus (23).

8. 8. The method according to any one of claims 1 to 7, wherein at least one morphological parameter of the nucleus (23) of the cell (22) is selected from circularity, roundness, solidity, aspect ratio, elliptical Fourier coefficients, and / or nuclear torsion.

9. 9. The method according to any one of claims 1 to 8, wherein for the distribution of a plurality of morphological parameters or each morphological parameter, morphological parameters are selected such that a statistical difference is established between test samples with different biological characteristics, in particular healthy and diseased.

10. Based on the fluorescence intensity profile and at least one morphological parameter determined for each nucleus (23), a) freely suspended nuclei, b) a deformed nucleus that extends at least partially into at least two adjacent microgrooves; c) trapped nuclei 10. The method according to claim 1, further comprising determining a deformation class of the nucleus (23) at a depth of one or more microgrooves (18) from at least three predetermined deformation classes corresponding respectively to:

11. 11. The method according to claim 1, comprising the steps of: determining the proportion of cells (22) in the cell sample that fall into at least one of the deformation classes, in particular the class of trapped nuclei and / or deformed nuclei; and determining the biological properties of the cell sample by comparing the proportion of cells (22) in the cell sample that fall into a plurality of deformation classes or each deformation class with a predetermined reference proportion of said at least one deformation class from a test sample of the same cell type, in order to determine the biological properties of the cell sample, wherein the biological properties of the test sample are known.

12. 12. The method according to claim 1, comprising determining a statistical distribution of at least one morphological parameter or a plurality of morphological parameters for nuclei of cells from a sample classified into one of the deformation classes, in particular the class of deformed nuclei and / or the class of trapped nuclei, and comparing a variable of said statistical distribution with the same variable of a statistical distribution of morphological parameters for cells classified into the same deformation class in a reference sample whose biological properties are known.

13. 1. A method for diagnosing a disease state in an individual, comprising at least the following steps: a) culturing a sample of a cell type isolated from said individual, wherein cells of the sample include somas and nuclei, and wherein the cells are cultured in a microstructured plate having a plurality of microgrooves on its surface, at least a portion of the surface of the microgrooves being an adhesive surface for the cells, the microgrooves being of a predetermined width and depth to allow at least partial engagement of the nucleus of at least one of said cells in one or more of the microgrooves; b) measuring by microscopy the fluorescent signal of the nuclei of cells from the sample, the nuclei of the cells being pre-configured to emit fluorescent radiation; c) determining a fluorescence intensity profile for each nucleus along at least one axis of said nucleus and at least one corresponding morphological parameter of said nucleus based on the fluorescence signal measured for each nucleus; d) determining a deformation class of each nucleus at the depth of one or more microgrooves based on the fluorescence intensity profile and at least one morphological parameter determined for said nucleus; e) comparing at least one characteristic of at least one variant class of nuclei of cells from the sample obtained in step d) with the same characteristic of a class of test samples in order to reach a conclusion therefrom regarding the diseased or non-disease state of the cells. A method comprising:

14. 1. A method for screening candidate compounds for the treatment and / or prevention of a disease state, comprising at least the following steps: a) in vitro culturing of a first sample of a cell type representative of a disease in the absence of a candidate compound; b) in vitro culturing, in the presence of a candidate compound, of a second sample of said cell type representative of said disease, wherein the cells of the first and second samples comprise somas and nuclei and are cultured in first and second identical microstructured plates, each having a plurality of microgrooves on its surface, at least a portion of the surface of the microgrooves being an adhesive surface for said cells, the microgrooves being of a predetermined width and depth to allow at least partial engagement of the nucleus of at least one of said cells in one or more of the microgrooves; c) measuring by microscopy the fluorescent signals of the nuclei of the cells from the first and second samples, wherein the nuclei of the cells from the first and second samples are pre-configured to emit fluorescent radiation; d) determining a fluorescence intensity profile along at least one axis of the nucleus and at least one morphological parameter of the nucleus for each cell from the first and second samples based on each measured fluorescence signal; e) determining a deformation class of the nucleus of each cell from the first and second samples in the direction of the depth of the microchannel based on the determined fluorescence intensity profile and the at least one determined morphological parameter; and f) comparing at least one characteristic of the first sample with the same characteristic for the second sample in at least one variant class of nuclei of cells from the first sample, wherein observing a difference between said characteristics of the first and second samples indicates the effectiveness of the candidate compound against said disease. A method comprising:

15. 15. The method of claim 14, comprising the steps of: culturing a third sample of the cell type believed to be healthy in the absence of the candidate compound; measuring fluorescent signals of nuclei of cells from the third sample by microscopy, wherein the nuclei of the cells of the third sample are pre-configured to emit fluorescent radiation; determining a fluorescent intensity profile along at least one axis of the nucleus and at least one morphological parameter of the nucleus for each cell from the third sample based on each measured fluorescent signal; determining a deformation class of the nucleus of each cell from the third sample in the direction of the depth of the microgroove based on the determined fluorescent intensity profile and the at least one determined morphological parameter; and comparing at least one characteristic of the first sample and / or the second sample in at least one deformation class of nuclei of cells from the first sample and / or the second sample with the same characteristic for the third sample, wherein observing a difference between the characteristics of the first and third samples and / or a similarity between the characteristics of the second and third samples indicates the effectiveness of the candidate compound for the disease.

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