Method for characterising the deformability of cells or a portion of cells in a cell sample

Abstract

The invention relates to a method for characterising the deformability of cells or a portion of cells in a sell sample, the method comprising: • - culturing the cells on 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, at least one portion of the surface of the microgrooves being a cell adhesion surface; • - measuring a fluorescence signal of the nuclei, which nuclei are pre-treated so as to emit fluorescence radiation; • - on the basis of the fluorescence signal measured for each nucleus, determining a fluorescence intensity profile and at least one morphological parameter of the nucleus; • - on the basis of the fluorescence intensity profile and the at least one morphological parameter, determining a deformation class of the nucleus in the depth of the microgrooves.

Description

[0001] Description

[0002] Title: Method for characterizing the deformability of cells or part of the cells in a cell sample

[0003] The present invention relates to a method for characterizing the deformability of cells or a portion of cells, in particular cell nuclei, of a cell sample. The present invention also relates to a method for diagnosing a pathological condition in an individual and to a method for screening a candidate compound for the treatment and / or prevention of a pathological condition.

[0004] Technical field

[0005] In general, the mechanical properties of cell nuclei are now considered important biomarkers in many pathologies. The most commonly used experimental systems in the laboratory to test these mechanical properties, such as atomic force microscopy, micropipette aspiration, or microrheometry, are low-throughput, technically complex, and / or costly in terms of equipment and time. New microfluidic experimental systems have been developed by circulating cells in channels. They allow higher throughputs, but remain complex.

[0006] H is known from the article Antmen E et al, Amplification of nuclear deformation of breast cancer cells by seeding on micropaterned 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 to deposit cancer cells whose nuclei are made fluorescent on a plate presenting protruding microreliefs in the form of parallel pillars smaller than the cells and to determine by fluorescence image analysis of the plate certain morphological parameters of the nuclei linked to their deformation on the plate due to the pillars to deduce whether the cells are metastatic or healthy. Such fluorescence image analysis is complex due to the diversity of shapes that cell nuclei can take on such a plate.

[0007] It is known from the article 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 to deposit cancer cells with fluorescent nuclei on a plate with parallel microgrooves smaller than the cells and to observe the morphology in the plane of the plate and the orientation of the cells and their nuclei in the direction of the microgrooves to deduce whether the cells are metastatic or healthy. Such an observation is made by fitting a contour ellipse for each core and determining its long and short axes, area, eccentricity and orientation in the microgroove for a very precise plate topology that must be determined precisely upstream. On the other hand, no deformation information in the depth is studied.

[0008] There is a need for a simple, robust and high-throughput method for characterizing the mechanical properties of cell nuclei deformation that is reliable, easy to implement in clinical practice and inexpensive, in particular to enable the performance of rapid and reliable functional tests making it possible in particular to detect a pathological or non-pathological state of a cell sample, for example with a view to establishing a diagnosis or screening a compound of interest.

[0009] Statement of the invention

[0010] The invention meets this need by a method for characterizing the deformability of cells or of a part of cells of a cell sample, said cells each comprising a body and a nucleus, the method comprising: the culture of said cells on a micro-structured plate having on the surface a plurality of microgrooves, the microgrooves being of predetermined width, spacing and depth so as to allow the at least partial engagement of the nucleus of at least one of said cells in one or more of the microgrooves, at least a part of the surface of the microgrooves being an adhesion surface for the cells,

[0011] The measurement by microscopy of a fluorescence signal from the nucleus of said cells, the nucleus of said cells being previously treated to emit fluorescence radiation, from the fluorescence signal measured for each nucleus, the determination of a fluorescence intensity profile for each nucleus along at least one axis of said nucleus and at least one morphological parameter of said nucleus, from the fluorescence intensity profile and at least one morphological parameter determined for each nucleus, the determination of a deformation class of said nucleus in the depth of one or more microgrooves.

[0012] As mentioned previously, cell nuclei can deform on a microstructured adherent surface. The deformation of nuclei depends on mechanical and biological characteristics of the cell, for example, its rigidity or its contractility. Such deformation characteristics can be different depending on the physiological or pathological state of a cell type. The study of the deformation characteristics of cell nuclei in a sample can therefore make it possible to determine a biological state, particularly a pathological state, of a cell sample.

[0013] The presence of microgrooves on the microstructured plate with an adhesion surface allows to generate on the cells a particular deformation stress controlled by the dimensions of the microgrooves on the plate. Indeed, the adhesion of the cells on the walls of the microgrooves causes the deformation of the nuclei, according to several known geometries and / or configurations, which can go as far as the complete trapping of the nuclei in the depth of the microgrooves. This allows to determine different deformation classes of the nuclei. Determining a deformation class for each nucleus can allow to compare the percentages of cells classified in the deformation classes with respect to a reference sample to deduce a biological characteristic.

[0014] The comparative study of a statistical intensity or morphometric variable, in particular of a distribution of a parameter of the fluorescence intensity profile or of a distribution of a morphological parameter, of the nuclei of the cells of the sample classified in at least one deformation class can allow for greater reliability in the determination of a biological characteristic of the sample than the comparative study of said statistical intensity or morphometric variable for all the nuclei of the cells of the sample.

[0015] The process is an ex vivo or extemporaneous process.

[0016] The process is not therapeutic as such.

[0017] Microstructured plate

[0018] Preferably, the microstructured plate comprises a transparent support, in particular made of glass, and a microstructured polymer structure having microgrooves on the surface, in particular made of polydimethylsiloxane (PDMS).

[0019] Preferably, the microgrooves are parallel to each other. Preferably, the microgrooves are of predetermined widths and depths so as to cause a deformation of a portion of the cells in the direction of the depth of the microgrooves.

[0020] The width, depth and spacing of the microgrooves may be selected based on at least one physical parameter of at least some cells of the cell type, including the size of said cells, the size of the nucleus of said cells and / or a biophysical characteristic of said cells, including the stiffness, adhesion strength or contractility of said cells. The width, depth and spacing of the microgrooves may be selected to have an average percentage of nuclei completely trapped in the microgrooves based on the cell type of the predetermined sample.

[0021] The average percentage of nuclei of healthy or abnormal cells of said cell type trapped entirely in the microgrooves can be between 5% and 95%, better between 10% and 90%.

[0022] Preferably, the width, spacing and depth of the microgrooves are between 30 and 60% of the length of the minor mean axis of the nucleus of the undeformed cells.

[0023] By "mean minor axis length" is meant the average length of the minor axis of an ellipse fitted to the shape of the nucleus of undeformed cells.

[0024] The width of the microgrooves is predetermined for a cell type and varies depending on the biological characteristics of the cell types studied. Generally, the width is greater than or equal to 3 pm and / or less than or equal to 10 pm.

[0025] The depth of the microgrooves is predetermined for a cell type and varies depending on the biological characteristics of the cell types studied. Generally, the depth is greater than or equal to 4 pm and / or less than or equal to 10 pm. For muscle cells, the depth can be between 4 and 5 pm in order to obtain a sufficiently high rate of nuclei in the microgrooves. In the case of breast epithelial cells, the depth can be between 7 and 9 pm, for example approximately equal to 8 pm. The microgrooves can all be of approximately constant depth over their entire length.

[0026] The microgrooves may have a spacing between them measured between adjacent edges greater than or equal to 3 pm and / or less than or equal to 10 pm. The microgrooves may all be of substantially constant width along their entire length. The microgrooves may all be of substantially constant depth along their entire length.

[0027] The microgrooves may all be of substantially constant spacing along their entire length.

[0028] Alternatively, the micro-structured plate may comprise distinct zones each comprising microgrooves of different width and / or spacing.

[0029] The depth can be fixed or variable on a microstructured plate and / or along a microgroove.

[0030] At least a portion of the inner surface of the microgrooves, in particular the side walls and / or the bottom of the microgrooves, preferably the entire surface of the microstructured plate, may be coated with an adhesion coating, in particular a cell adhesion protein, for example fibronectin, collagen, laminin or gelatin.

[0031] Cell sample

[0032] The cells may be adherent cells, for example muscle cells, endothelial cells, stem cells, preferably non-embryonic or non-human, epithelial cells, nerve cells, bone cells, fat cells, podocytes, cancer cells or a mixture of such cells.

[0033] The method may include a step of fixing the cells on the microstructured plate by adding an alcohol compound, in particular methanol, or an aldehyde compound, in particular paraformaldehyde. The step of fixing the cells may be carried out at least one hour after the sample has been deposited on the microstructured plate. Such a time allows the cells time to deform before fixing in order to have a significant result of the deformation of the cells in the sample.

[0034] The method may include treating the cell sample so that the nuclei emit the aforementioned fluorescent radiation. This step may take place before or after placing the sample cells on the microstructured plate, preferably after placing the sample cells on the microstructured plate, more preferably after fixing the cells on the microstructured plate. The nucleus of the sample cells may be labeled before or after fixing with a fluorescent nuclear marker, including a Hoechst dye or DAPI.

[0035] Alternatively, treating 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.

[0036] The cell sample can be of human, animal or plant origin.

[0037] Preferably, the cell sample is configured so that the cell density of the cell sample at the time of measurement by microscopy avoids cell confluence, in particular such that the cell confluence is less than or equal to 60%. For example, for muscle, endothelial or epithelial cells, the cell density of the sample deposited on the microstructured plate may be greater than or equal to 10,000 cells / cm 2 and / or less than or equal to 50,000 cells / cm 2

[0038] Measurement by microscopy

[0039] The measurement by microscopy may involve the acquisition of a fluorescence image of the surface of the microstructured plate. The fluorescence image obtained may be a view from above or below by transparency of the microstructured plate, in particular obtained by epifluorescence.

[0040] Alternatively or in combination, the measurement by microscopy may comprise the acquisition of fluorescence images in a plane or a plurality of planes transverse to the microgrooves, in particular by confocal microscopy.

[0041] The measurement by microscopy may include, on the fluorescence image(s) acquired, the detection of the fluorescence signal emitted by the nucleus of each cell.

[0042] Image analysis

[0043] The method, in particular the step of determining the fluorescence intensity profile and the morphological parameter(s), may comprise the detection of the nuclei on the image as well as their outline from the fluorescence signal emitted by the nucleus, in particular visible on the acquired fluorescence image(s), and the morphometric analysis of said shape of the nucleus.

[0044] All of the steps for determining the fluorescence intensity profile and at least one morphological parameter and determining the deformation class can be carried out automatically, in particular by a processor implementing software for processing the fluorescence images obtained by microscopy measurement. The processor may include machine or deep learning software.

[0045] Determination of fluorescence intensity profile

[0046] The fluorescence intensity profile of each nucleus can be determined from the fluorescence signal, in particular on the acquired fluorescence image, perpendicular to the extension axis of the microgrooves, preferably in a substantially median plane of said nucleus.

[0047] Determination of morphological parameter

[0048] The at least one morphological parameter of the cell nucleus may be selected from circularity, roundness, solidity, aspect ratio and / or Fourier elliptic coefficient ratio of the nucleus and / or negative mean curvature of the nucleus contour.

[0049] By "negative mean curvature" we mean the average of the concave curvatures of the core contour.

[0050] The morphological parameter(s) may be chosen, in particular by prior analyses on reference samples, so as to be one or more relevant parameters for discriminating between different biological characteristics of the sample, in particular between a pathological characteristic and a healthy characteristic. The morphological parameter(s) are chosen so that, for a distribution of the or each morphological parameter, a statistical difference is established between samples having different biological characteristics, in particular healthy and pathological,

[0051] Determination of the deformation class

[0052] Preferably, the method comprises, from the fluorescence intensity profile and at least one morphological parameter for each nucleus, determining the deformation class of said nucleus in the depth of one or more microgrooves from among at least three predetermined deformation classes corresponding respectively to: a) a free nucleus in suspension, corresponding for example to a substantially flat fluorescence intensity profile or without a major fluorescence intensity peak associated with one or more morphological parameters characteristic of a slightly deformed nucleus contour, in particular substantially circular, and / or reduced tortuosity of the nucleus contour, for example circularity, roundness, solidity and / or a high Fourier elliptic coefficient ratio, in particular greater than or equal to a respective predetermined threshold value, and / or a low aspect ratio and / or negative mean curvature,in particular less than or equal to a respective predetermined threshold value, b) a deformed core extending in at least two adjacent microgrooves, corresponding for example to a fluorescence intensity profile having at least two main peaks associated with at least one morphological parameter characterizing a significant tortuosity of the contour, in particular a low solidity compared to a predetermined threshold value and / or a low Fourier elliptic coefficient ratio compared to a predetermined threshold value and / or a high negative mean curvature compared to a predetermined threshold value, and / or a low circularity and / or roundness compared to a predetermined threshold value, c) a trapped core, in particular totally inserted in a microgroove, corresponding for example to a fluorescence intensity profile having a main peak associated with at least one morphological parameter characterizing an elongation of the core,in particular a low circularity and / or roundness compared to a predetermined threshold value, and / or a high aspect ratio compared to a predetermined threshold value e,

[0053] The method may comprise comparing at least one statistical characteristic of at least one deformation class of the nuclei of the cells of the sample obtained with the same characteristic for a reference sample. The statistical characteristic may be the proportion of cells of the cell sample in at least one deformation class and / or a statistical or morphometric variable of the nuclei of the cells classified in at least one deformation class.

[0054] The method may comprise determining the proportion of cells in the cell sample in at least one of the deformation classes, in particular the class of trapped and / or deformed nuclei. The method may comprise determining a biological characteristic of the cell sample by comparing the proportion of cells in the cell sample in the or each deformation class, with a proportion in said at least one deformation class of the cells in a reference sample of the same cell type and whose biological characteristic is known. The method may comprise determining a statistical distribution of the or at least one of the morphological parameters of the nuclei of the cells in the sample classified in at least one of the deformation classes, in particular for the class of deformed nuclei and / or that of trapped nuclei.The method then preferably comprises the comparison of a variable of said statistical distribution of the sample with the same variable of the statistical distribution of the morphological parameter of cells classified in said same deformation class(es) of a reference sample whose biological characteristic is known. The inventors have demonstrated that limiting the morphological comparison to cells of a particular deformation class of the sample makes it possible to have a more precise biological characterization of the sample than by taking the cells of the entire sample.

[0055] The biological characteristic can be the pathological or non-pathological nature of the cell sample.

[0056] The method may include a prior step of determining the deformation class whose proportion of cells classified in said deformation class measured for reference samples allows the best possible discrimination of at least two populations of different biological characteristics,

[0057] Alternatively or additionally, the method may comprise a prior step of determining a combination of one or more morphological parameters of the nuclei and one or more deformation classes for which a variable of the statistical distribution of the morphological parameter(s) of the nuclei classified in the deformation class(es) allows the best possible discrimination of two reference populations with different biological characteristics,

[0058] This or these preliminary determination steps may include one or more statistical tests for comparing the samples.

[0059] Diagnostic method

[0060] The invention also meets this need by a method for diagnosing a pathological condition in an individual, the method comprising at least the following steps: a) Culturing a sample of a type of cells isolated from said individual, the cells of the sample comprising a body and a nucleus and being cultured on a micro-structured plate having on the surface a plurality of microgrooves, at least a portion of the surface of the microgrooves being an adhesion surface for the cells, the microgrooves being of predetermined width and depth so as 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 a fluorescence signal of the nuclei of the cells of the sample, the nuclei of the cells being previously configured to emit fluorescence radiation, c) from the fluorescence signal measured for each nucleus,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, d) from the fluorescence intensity profile and at least one morphological parameter determined for each nucleus, determining a deformation class of said nucleus in the depth of one or more microgrooves, e) Comparing at least one characteristic of at least one deformation class of the nuclei of the cells of the sample obtained in step d) with the same characteristic for a reference sample to conclude on the pathological state of the individual.,

[0061] The characteristics described above in relation to the method for characterizing the deformability of a cell or part of a cell apply to this diagnostic method in combination or independently of each other and independently of the method for characterizing the deformability of a cell or part of a cell.

[0062] Screening method

[0063] The invention also meets this need by a method for screening a candidate compound for the treatment and / or prevention of a pathological condition, the method comprising at least the following steps: a) The in vitro culture of a first sample of a cell type representative of a pathology in the absence of the candidate compound, b) The in vitro culture of a second sample of said cell type representative of said pathology in the presence of the candidate compound, the cells of the first and second samples comprising a body and a nucleus and being cultured on a first and a second identical micro-structured plate each having on the surface a plurality of microgrooves, at least a portion of the surface of the microgrooves being an adhesion surface of said cells,the microgrooves being of predetermined width and depth so as to allow at least partial engagement of the nucleus of at least one of said cells in one or more of the microgrooves, c) The measurement by microscopy of a fluorescence signal of the nuclei of the cells of the first and second samples, the nuclei of the cells of the first and second samples being previously configured to emit fluorescence radiation, d) The determination of fluorescence intensity profiles along at least one axis of the nucleus and of at least one morphological parameter of the nucleus of each cell of the first and second samples from the respective fluorescence signals measured, e) The determination of deformation classes of the nucleus of each cell of the first and second samples in the direction of the depth of the microgrooves from the determined fluorescence intensity profile and at least one determined morphological parameter,and f) Comparing at least one characteristic of the first sample in at least one class of deformation of the nuclei of the cells of the first sample with the same characteristic for the second sample, the observation of a difference between said characteristics of the first and second samples being indicative of an efficacy of the candidate compound with regard to said pathology.,

[0064] The method may comprise culturing a third sample of said cell type considered healthy in the absence of the candidate compound, measuring by microscopy a fluorescence signal of the nuclei of the cells of the third sample, the nuclei of the cells of the third sample being previously configured to emit fluorescence radiation, determining the fluorescence intensity profile along at least one axis of the nucleus and at least one morphological parameter of the nucleus of each cell of the third sample from the respective fluorescence signals measured, determining deformation classes of the nucleus of each cell of the third sample in the direction of the depth of the microgrooves from the determined fluorescence intensity profile and 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 class of deformation of the nuclei of the cells of the first sample and / or the second sample with the same characteristic for the third sample, the observation of a difference between said characteristics of the first and third samples and / or of a similarity between said characteristics of the second and third samples being indicative of an efficacy of the candidate compound with regard to said pathology.,

[0065] The characteristics described above in relation to the method for characterizing the deformability of a cell or part of a cell apply to this screening method in combination or independently of each other and independently of the method for characterizing the deformability of a cell or part of a cell.

[0066] Preferably, the characteristic being the proportion of cells of the samples in at least one of the deformation classes, in particular the class of trapped nuclei, the observation of a statistical difference between the first and the second sample, and / or the observation of a non-significant statistical difference between the healthy sample and the sample in the presence of the candidate compound and significant between the healthy sample and the sample without candidate compound, being indicative of an efficacy of the candidate compound with regard to said pathology.

[0067] The characteristic being, in one of the deformation classes, in particular in the class of deformed nuclei or preferentially in the class of trapped nuclei, the statistical distribution of the or at least one of the morphological parameters of the cells of the first and second samples. The method then preferentially comprises the comparison of said statistical distribution of the first sample with a statistical distribution profile of the second sample, the observation of a statistical difference between the first and second samples, and / or the observation of a non-significant statistical difference between the healthy sample and the sample in the presence of the candidate compound and significant between the healthy sample and the sample without candidate compound, being indicative of an efficacy of the candidate compound with regard to said pathology.

[0068] Brief description of the drawings

[0069] [Fig 1] schematically represents the overall process of characterizing the deformability of cells or a part of a cell in a cell sample

[0070] [Fig 2A] shows a detail of a microstructured plate in section carrying cells whose nuclei are trapped in the microgrooves,

[0071] [Fig 2B] represents a microstructured plate carrying cells whose nuclei are visible in fluorescence in perspective and in section along AA, [Fig 2C] represents a microstructured plate carrying cells whose bodies are visible in fluorescence in perspective and in section along AA,

[0072] [Fig 3] represents fluorescence images of nuclei acquired for samples of different cell types,

[0073] [Fig 4] is a graph showing the percentage of trapped nuclei identified as a function of mean nuclear volume for some of the cell types in Figure 3,

[0074] [Fig 5] represents the deformation classes and an example of fluorescence intensity profile corresponding to each deformation class,

[0075] [Fig 6] is an acquired fluorescence image on which the nuclei of each deformation class have been identified in different colors,

[0076] [Fig 7] represents fluorescence images of myoblast nuclei acquired for microgrooves of different widths,

[0077] [Fig 8] is a box plot representing the percentage of trapped nuclei as a function of the widths in Figure 7,

[0078] [Fig 9] is a box plot representing the percentage of deformed nuclei as a function of the widths in Figure 7,

[0079] [Fig 10] represents fluorescence images acquired for plates of different depths,

[0080] [Fig 11] is a box plot of the percentage of trapped nuclei as a function of depths in Figure 10,

[0081] [Fig 12] is a box plot of the percentage of deformed cores as a function of depths in Figure 10,

[0082] [Fig 13] is a box plot representing the percentage of trapped nuclei for a healthy (WT) and a pathological (MU Lamin A) sample of myoblasts,

[0083] [Fig 14] is a box plot representing the percentage of deformed nuclei for a healthy (WT) and a pathological (MU Lamin A) sample of myoblasts,

[0084] [Fig 15] is a box plot representing the percentage of trapped nuclei for a healthy sample (MCF10A) and a pathological sample (MCF7) of breast epithelial cells, [Fig 16] represents the statistical distributions of circularity determined on all cells in the sample (a), cells with trapped nuclei (b) and cells with distorted nuclei (c) for a healthy sample (WT) and a pathological sample (MU) of myoblasts, and

[0085] [Fig 17] represents the statistical distributions of the ratio of the elliptic Fourier coefficients determined on all the cells in the sample (a), the cells with trapped nuclei (b) and the cells with deformed nuclei (c) for a healthy sample (WT) and a pathological sample (MU) of myoblasts.

[0086] Detailed description

[0087] Figure 1 illustrates the entire process for characterizing the deformability of cells or a part of a cell in a cell sample.

[0088] The cell sample may be a sample of adherent cells comprising muscle cells, endothelial cells, epithelial cells, nerve cells, bone cells, fat cells, podocytes, cancer cells, or a mixture of said cells. The cells may be human, animal, or plant.

[0089] In step 10, the cell sample is deposited on the surface of a micro-structured plate 12, illustrated in FIGS. 2A to 2C, and is cultured on the surface of the plate 12 in a suitable medium for a period of at least 1 h.

[0090] The plate 12 comprises a transparent support 14, in particular made of glass, and a micro-structured structure 16 made of polymer, in particular made of polydimethylsiloxane (PDMS), secured to the support. The micro-structured structure 16 has on the surface a plurality of micro-grooves 18, preferably of width l and depth p constant over their entire length and identical, parallel and regularly spaced apart. But it could be otherwise, the micro-grooves could comprise at least two distinct longitudinal parts each of constant width and depth but which differ from each other by their depth and / or their width, and / or the micro-structured plate could comprise at least two distinct zones each comprising identical micro-grooves in said zone and which differ between the two zones by the width, depth and / or spacing of the micro-grooves.

[0091] The microgrooves 18 are of predetermined width / and depth p so as to allow at least partial engagement of the nucleus 23 of at least one of said cells in one or more of the microgrooves. They are also dimensioned, preferably, to prevent the entire body of said cells 22 from being engaged in a microgroove 18. To do this, the width l and the depth p of the microgrooves 18 can be chosen as a function of at least one physical parameter of cells of the type of those in the sample, in particular their average dimension or the average dimension of their nucleus, and / or a biophysical characteristic of these cells, in particular their average rigidity, the average adhesion force or the average contractility.The width l and the depth p of the microgrooves 18 can be chosen so that the percentage of nuclei completely trapped in the microgrooves 18 is a significant parameter of a biological characteristic of the cells of the cell type that one seeks to determine, in particular of the pathological or non-pathological nature of the cells. For example, the average percentage of nuclei of healthy or malignant cells of said cell type, in particular in the case of endothelial or muscle cells, trapped entirely in the microgrooves can be between 10% and 90%. The width / , the spacing e and the depth p of the microgrooves 18 can be between 30 and 60% of the length of the minor mean axis of the nucleus of the undeformed cells.The width / of the microgrooves may be between 3 and 10 pm, the depth p of the microgrooves may be between 4 and 10 pm and the spacing e between them measured between adjacent edges of the microgrooves may be between 3 and 10 pm.

[0092] The surface of the microstructured structure 16 is at least partly treated with a cell adhesion agent, such as fibronectin 20. Such treatment can be done by passing through plasma then incubating in a solution containing fibronectin. The treatment is preferably done over the entire surface of the plate 12 but it could be otherwise. It could be done only in the bottom of the microgrooves 18 and / or on the side walls.

[0093] When culturing the cells 22, the latter deform or not due to the reliefs of the plate 12 and the nuclei 23 of the cells engage more or less in the microgrooves of the plate 12.

[0094] The cells 22 are fixed by adding a fixing compound, for example an aldehyde compound, especially paraformaldehyde and the cell nuclei are labeled with a fluorescent marker such as 4',6-diamidino-2-phenylindole (DAPI) after permeabilization of the cells with a permeabilizing agent such as Triton X-100. It could be labeled differently, in particular by transducing the cells with a plasmid encoding a nuclear protein fused to a fluorescent protein, or by adding a fluorescent nuclear marker before fixation, such as Hoechst dyes. Fixing the cells makes it possible to overcome their dynamics because the confinement of the nuclei in the microgrooves is statistically reversible over time.

[0095] Preferably, the cell sample is configured so that the cell density of the cell sample at the time of their fixation avoids cell confluence. The cell density at the time of cell fixation may be between 10,000 and 50,000 cells / cm 2 .

[0096] Fluorescence images of the surface of the plate 12 are then taken at step 30 using a fluorescence microscope. As illustrated in Figure 3, the nuclei of the cells and their shapes are identifiable by the fluorescence they emit. It is clear from Figure 3 that this is applicable to the various cell types mentioned above.

[0097] The analysis, preferably automatic, 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 longitudinal axis X of the microgrooves and substantially median to the nucleus.

[0098] This also makes it possible to determine for each core 23 a contour, in particular by adjusting a shape model, for example an ellipse model, to deduce different shape parameters of the cores, in particular the following parameters:

[0099] Roundness is defined by the ratio of the area of ​​the core to the area of ​​a circle yj circumscribed to the core, which gives the following formula: 4 * A being the area of ​​the core and M being the greatest length of the core

[0100] Circularity is defined by the ratio of the area of ​​the core to the area of ​​a circle whose radius is defined by the length of the contour of the core, which gives the following formula yj: 4TT * — C being the length of the contour of the core,

[0101] The aspect ratio is defined by the ratio of the length of the largest axis to the length of the smallest axis of an ellipse model,

[0102] Solidity is defined as the ratio of the area of ​​the core to the area of ​​the convex hull of the core,

[0103] The ratio of the Fourier elliptic coefficients is defined by the following formula: being the length of the major axis of the harmonic ellipse model n and m n being the length of the minor axis of the n-harmonic ellipse model, and / or

[0104] Negative mean curvature is defined as the average of the concave curvatures of the core contour.

[0105] The fluorescence intensity profile and the morphological parameter(s) make it possible, in step 50, to classify each cell whose nucleus 23 is identifiable into a deformation class from among several pre-established deformation classes. The deformation classes may in particular be a function of the percentage of penetration of the nucleus into the microgrooves. The classification is done, for example, among three classes corresponding respectively to: i) a free nucleus in suspension, corresponding for example to a substantially flat fluorescence intensity profile or one without a major fluorescence intensity peak associated with one or more morphological parameters characteristic of a slightly deformed nucleus contour, in particular substantially circular, and / or reduced tortuosity of the nucleus contour, for example circularity, roundness, solidity and / or a high Fourier elliptic coefficient ratio, in particular close to 1,and / or an aspect ratio and / or a low negative mean curvature, in particular less than or equal to a respective predetermined threshold value, ii) a deformed core extending into at least two adjacent microgrooves, corresponding for example to a fluorescence intensity profile having at least two main peaks associated with at least one morphological parameter characterizing a significant tortuosity of the contour, in particular a low solidity compared to a predetermined threshold value and / or a low Fourier elliptic coefficient ratio compared to a predetermined threshold value and / or a high negative mean curvature compared to a predetermined threshold value, and / or a low circularity and / or roundness compared to a predetermined threshold value, iii) a trapped core, in particular totally inserted in a microgroove,corresponding for example to a fluorescence intensity profile presenting a main peak associated with at least one morphological parameter characterizing an elongation of the nucleus, in particular a low circularity and / or roundness compared to a predetermined threshold value, and / or a high aspect ratio compared to a predetermined threshold value. Figure 5 represents for each of the 3 classes above an example of a fluorescence intensity profile. As can be seen in Figure 5, it is also possible to help with the classification to make a confocal measurement for each nucleus, in particular in a median plane of the nucleus transverse to the plate and perpendicular to the grooves. Such a measurement allows the clear visualization of the penetration depth of the nucleus, which can allow the classification to be refined, in particular during a preliminary study of the relevant parameters. However, such a measurement is not essential.,

[0106] The class of each nucleus can be reported on the fluorescence image by a color code to allow rapid identification directly on the image, as illustrated in Figure 6.

[0107] There are then several possibilities for determining a biological characteristic of the cell sample, in particular its pathological or healthy character, at step 60. The percentage of cells in the sample in one of the classes, in particular the class of trapped nuclei or deformed nuclei, can be compared with that of another reference sample whose biological characteristic is known, in particular a healthy sample, as can be seen in Figures 13 to 15. It is also possible to compare, in one of the deformation classes, in particular the class of trapped nuclei or that of deformed nuclei, a statistical distribution of one of the morphological parameters with that of a test sample of cells whose biological characteristic is known, in particular a healthy sample, as can be seen in Figures 16 or 17. These different methods can be combined with each other if necessary.The method for determining the biological characteristic depends in particular on the plate used, the cell type and the cell characteristic to be determined. The best discrimination method can be determined beforehand by statistical tests. These statistical tests are carried out under the same general conditions (same type of plate, same culture process and same imaging method) on samples with different known biological characteristics, in particular pathological and healthy. These preliminary statistical tests are preferably carried out on several panels of reference samples in order to verify the stability of the identification method.

[0108] Then, depending on the comparison obtained between the sample to be tested and the reference sample, either it is identified that the biological characteristic is identical to that of the reference population in step 70, or it is identified that the biological characteristic is different from that of a reference population in step 80.

[0109] Such a study has several possible applications. For example, it can be used to determine whether a patient sample is pathological or not by comparing it to one or more healthy or pathological reference populations in order to deduce a diagnosis. It can also be used to screen a candidate compound by comparing the result on a sample having received the candidate compound with that for a control sample not having received it and optionally by comparing the result of the sample having received the candidate compound and the control sample not having received it to that of a reference population, in particular a population considered healthy.

[0110] Example 1

[0111] Figures 3 and 4 illustrate a study of different cell types.

[0112] Figure 3 shows the epifluorescence imaging images on top and confocal Z-slice imaging images on the bottom for the following cell types: a) myoblasts, b) human umbilical vein endothelial cells (HUVECs), c) COS-7 cells, d) HeLa cells, e) parietal epithelial cells (PECs), f) breast epithelial cells (MCF-10A), g) breast cancer cells (MDA-MB-231), h) breast cancer cells (MCF7), and i) podocytes.

[0113] Cells are cultured on a microstructured plate having parallel microgrooves 5 pm wide and deep, regularly spaced 5 pm apart.

[0114] These images show that all the cells studied are capable of deforming on the plate. The degree of deformation of the nuclei depends on the cell type.

[0115] Figure 4 shows for some of these cells the percentage of nuclei in the cell sample belonging to the class of trapped nuclei P p as a function of the average nuclear volume V m cells (measured on a flat surface in pm) for four of the above-mentioned cell types. It is noted that the percentage of trapped nuclei in the P samples p is not directly dependent on the mean nuclear volume V m . Which explains why there is no simple link between the volume of the nuclei and their deformation.

[0116] Example 2

[0117] Figures 7 to 12 illustrate a study of the influence of microgroove dimensions on nuclei deformation for myoblast samples.

[0118] In this example, the plates are made of micro-structured PDMS on a glass support. They are covered over their entire surface with a fibronectin coating. Myoblast-type cell samples are cultured on the plate in a medium for 8 h. At the end of the culture, the cells are fixed with 4% paraformaldehyde for 15 min. After a permeabilization step with Triton, the nuclei are labeled with a fluorescent marker DAPI for 1 h. Images of the nuclei are then taken using a fluorescence microscope (x20 objective).

[0119] Figure 7 shows the images obtained by epifluorescence imaging for myoblast samples on supports having microgrooves spaced 5 pm apart, 4 pm deep and of different widths. The widths are as follows: a) 1 = 3 pm, b) l = 5 pm, and c) l = 7 pm.

[0120] Figures 8 and 9 respectively represent the percentage of nuclei classified in the class of trapped nuclei P p and the percentage of nuclei classified in the class of deformed nuclei P a as a function of the width l of the microgrooves. It can be seen that increasing the width of the microgrooves changes the proportion of the different classes of nuclei (decrease in the percentage of deformed nuclei and increase in the percentage of trapped nuclei).

[0121] Figure 10 represents the images obtained by epifluorescence imaging for myoblast samples on supports having microgrooves spaced 5 pm apart, 5 pm wide and of different depths. The depths being as follows: a) p=4 pm, and b) p=5.4 pm, Figures 11 and 12 respectively represent the percentage of nuclei classified in the class of trapped nuclei P pand the percentage of nuclei classified in the class of deformed nuclei P a as a function of the depth p of the microgrooves. It is observed that increasing the depth of the microgrooves changes the proportion of the different classes of nuclei (decrease in the percentage of deformed nuclei and increase in the percentage of trapped nuclei).

[0122] Thus, the dimension of the microgrooves has a strong influence on the deformation of the cells on the microstructured plate. The dimensions of the microgrooves can therefore be adapted for each cell type according to their deformation potential, in particular by a preliminary study to optimize the study of cell deformation.

[0123] Example 3

[0124] Figures 13 and 14 respectively represent the percentages of trapped nuclei P pand the percentage of deformed nuclei P a for samples of myoblast-type cells from healthy patients (WT) and for samples of myoblast-type cells from sick patients with a mutation in the gene coding for lamin A, the main component of the nuclear envelope (Mu Lamin A Delta K32). The cell samples are cultured by the method described in the previous example on a plate with microgrooves of width 1 of 5 pm, depth of 4 pm and spaced 5 pm apart.

[0125] It is observed that the percentages of trapped nuclei P p and distorted P a between healthy WT samples and pathological Mu Lamin A samples with their minimum and maximum do not overlap, which indicates that a significant difference exists on these parameters between healthy WT samples and pathological Mu Lamin A samples. This result shows that the determination of the percentage of trapped nuclei P por deformed Pa allows the discrimination of WT and Mu Lamin A samples, and therefore the detection of pathology.

[0126] Example 4

[0127] Figure 15 represents the percentages of trapped nuclei P p for a sample of healthy epithelial breast cells (MCF10A) and for a sample of tumoral epithelial breast cells (MCF7). The cell samples are cultured for 24 hours using the method described in the previous example on a plate with microgrooves of width 1 of 5 pm, depth of 7.5 pm and spaced 5 pm apart. A significant difference in the percentage of trapped nuclei P is clearly observed p between the non-tumor cell sample MCF10A and the tumor cell sample MCF7. This result shows that the percentage of trapped nuclei is also a good discriminator in the case of breast epithelial cells.

[0128] Example 5

[0129] Figure 16 shows the normalized statistical distributions of circularity for all cells in the sample (a), trapped nuclei only (b), and deformed nuclei only (c) for a sample of myoblast-like cells from a healthy patient (WT) and for a sample of myoblast-like cells from a diseased patient with a Lamin A mutation (Mu). The cell samples are cultured by the method described in the previous example on a plate with microgrooves 5 pm wide, 4 pm deep, and spaced 5 pm apart.

[0130] The difference d between the maxima of the two WT and Mu distributions is calculated to determine the separation of the distributions and thus characterize the discrimination potential between the two cell samples. The difference d is equal to 0.06 in case (a) (all cells), 0.25 in case (b) (trapped nuclei), 0.23 in case (c) (deformed nuclei).

[0131] It is observed that the difference d is much greater in cases (b) and (c), which attests that the classification system prior to the morphological study of the nuclei improves the discrimination between the two WT and Mu cell samples.

[0132] Figure 17 represents the same study applied to the ratio of the elliptic Fourier coefficients. The difference d is equal to 0.12 in case (a) (all cells), 0.31 in case (b) (trapped nuclei), 0.12 in case (c) (deformed nuclei).

[0133] It is observed that the difference d is much greater in cases (b), which attests that the classification system prior to the morphological study of the nuclei improves the discrimination between the two WT and Mu cell samples.

[0134] This time, however, only the class of trapped nuclei allows for better discrimination. This shows that a preliminary study can be useful to determine the right combination of classes and morphological parameters to study in order to have optimal discrimination of samples with different biological characteristics.

[0135] The invention is not limited to the example just described. For example, the microstructured plate may have microgrooves in a more complex distribution than regularly spaced. The plate could include several areas having different microgroove characteristics.

[0136] Alternatively, other morphological parameters characterizing the shape of the nucleus in three dimensions are conceivable if they allow effective discrimination of two cell samples with different biological characteristics.

[0137] Alternatively, the invention is not limited to the cell types and pathology mentioned. The method mentioned has potential for many cell types and many pathologies.

Claims

Claims 1. Method for characterizing the deformability of cells (22) or of a portion of cells (22) of a cell sample, said cells (22) each comprising a body and a nucleus (23), the method comprising: culturing said cells (22) on a microstructured plate (12) having on its surface a plurality of microgrooves (18), the microgrooves (18) being of predetermined width l and depth p so as to allow at least partial engagement of the nucleus (23) of at least one of said cells (22) in one or more of the microgrooves (18), at least a portion of the surface of the microgrooves (18) being an adhesion surface for the cells, The measurement by microscopy of a fluorescence signal of the nucleus (23) of said cells (22), the nucleus (23) of said cells (22) being previously treated to emit fluorescence radiation, from the fluorescence signal measured for each nucleus (23), the determination of 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), from the fluorescence intensity profile and the at least one morphological parameter determined for each nucleus, the determination of a deformation class of said nucleus in the depth of one or more microgrooves (18).

2. 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 adhesion coating (20), in particular a cell adhesion protein, for example fibronectin, collagen, laminin or gelatin.

3. Method according to any one of the preceding claims, in which the cells (22) are chosen from adherent cells, such as for example muscle cells, endothelial cells, epithelial cells, podocytes and / or cancer cells.

4. Method according to any one of the preceding claims, in which the measurement by microscopy comprises the acquisition of a fluorescence image of the surface of the microstructured plate (12).

5. Method according to claim 4, in which the determination of the morphological parameter(s) may comprise the detection of the outline of the nucleus (23) of the cells (22) on the acquired fluorescence image(s) and the morphometric analysis of said shape of the nucleus (23) to deduce the morphological parameter(s).

6. Method according to any one of the preceding claims, in which the measurement by microscopy comprises the acquisition of fluorescence images in a plane and / or a plurality of planes transverse to the microgrooves (18), in particular by confocal microscopy.

7. Method according to any one of the preceding claims, in which the fluorescence intensity profile is determined on the fluorescence signal, in particular on the acquired fluorescence image, perpendicular to the axis of extension of the microgrooves (18), preferably in a substantially median plane of said core (23).

8. Method according to any one of the preceding claims, in which the at least one morphological parameter of the nucleus (23) of the cells (22) is chosen from circularity, roundness, solidity, aspect ratio, Fourier elliptical coefficient and / or tortuosity of the nucleus.

9. Method according to any one of the preceding claims, in which the morphological parameter(s) are chosen so that, for a distribution of the or each morphological parameter, a statistical difference is established between test samples having different biological characteristics, in particular healthy and pathological.

10. Method according to any one of the preceding claims, in which the method comprises, from the fluorescence intensity profile and the at least one morphological parameter determined for each nucleus (23), the determination of the deformation class of said nucleus (23) in the depth of one or more microgrooves (18) among at least three predetermined deformation classes corresponding respectively to: a) a free nucleus in suspension, b) a deformed nucleus extending at least partially in at least two adjacent microgrooves, c) a trapped nucleus.

11. Method according to any one of the preceding claims, comprising determining the proportion of cells (22) of the cell sample in at least one of the deformation classes, in particular the class of trapped and / or deformed nuclei and determining a biological characteristic of the cell sample by comparing the proportion of cells (22) of the cell sample in the or each deformation class, with a reference proportion in said at least one deformation class of a test sample of the same cell type determined beforehand to determine a biological characteristic of the cell sample, the biological characteristic of the test sample being known.

12. Method according to any one of the preceding claims, comprising the determination of a statistical distribution of the or at least one of the morphological parameters of the nuclei of the cells of the sample classified in one of the deformation classes, in particular for the class of deformed nuclei and / or that of trapped nuclei and the comparison of a variable of said statistical distribution with the same variable of the statistical distribution of the morphological parameter of cells classified in said same deformation class(es) of a reference sample of which a biological characteristic is known.

13. A method for diagnosing a pathological condition in an individual, the method comprising at least the following steps: a) Culturing a sample of a type of cells isolated from said individual, the cells of the sample comprising a body and a nucleus and being cultured on a micro-structured plate having on its surface a plurality of microgrooves, at least a portion of the surface of the microgrooves being an adhesion surface for the cells, the microgrooves being of predetermined width and depth so as 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 a fluorescence signal of the nuclei of the cells of the sample, the nuclei of the cells being previously configured to emit fluorescence radiation, c) from the fluorescence signal measured for each nucleus,determining a fluorescence intensity profile for each nucleus along at least one axis of said nucleus and at least one morphological parameter of said corresponding nucleus, d) from the fluorescence intensity profile and at least one morphological parameter determined for each nucleus, the determination of a deformation class of said nucleus in the depth of one or more microgrooves, e) The comparison of at least one characteristic of at least one deformation class of the nuclei of the cells of the sample obtained in step d) with the same characteristic for classes of a test sample to conclude on the pathological state or not of the cells.

14. Method for screening a candidate compound for the treatment and / or prevention of a pathological condition, the method comprising at least the following steps: a) The in vitro culture of a first sample of a cell type representative of a pathology in the absence of the candidate compound, b) The in vitro culture of a second sample of said cell type representative of said pathology in the presence of the candidate compound, the cells of the first and second samples comprising a body and a nucleus and being cultured on a first and a second identical micro-structured plate each having on the surface a plurality of microgrooves, at least a portion of the surface of the microgrooves being an adhesion surface of said cells, the microgrooves being of predetermined width and depth so as 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 a fluorescence signal of the nuclei of the cells of the first and second samples, the nuclei of the cells of the first and second samples being previously configured to emit fluorescence radiation, d) determining fluorescence intensity profiles along at least one axis of the nucleus and at least one morphological parameter of the nucleus of each cell of the first and second samples from the respective measured fluorescence signals, e) determining deformation classes of the nucleus of each cell of the first and second samples in the direction of the depth of the microgrooves from the determined fluorescence intensity profile and at least one determined morphological parameter, and f) comparing at least one characteristic of the first sample in at least one deformation class of the nuclei of the cells of the first sample with the same characteristic for the second sample,the observation of a difference between, said characteristics of the first and second samples being indicative of the efficacy of the candidate compound with regard to said pathology.

15. The method of claim 14, comprising culturing a third sample of said cell type considered healthy in the absence of the candidate compound, measuring by microscopy a fluorescence signal of the nuclei of the cells of the third sample, the nuclei of the cells of the third sample being previously configured to emit fluorescence radiation, determining the fluorescence intensity profile along at least one axis of the nucleus and at least one morphological parameter of the nucleus of each cell of the third sample from the respective fluorescence signals measured, determining deformation classes of the nucleus of each cell of the third sample in the direction of the depth of the microgrooves from the determined fluorescence intensity profile and 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 class of deformation of the nuclei of the cells of the first sample and / or the second sample with the same characteristic for the third sample, the observation of a difference between said characteristics of the first and third samples and / or of a similarity between said characteristics of the second and third samples being indicative of an efficacy of the candidate compound with regard to said pathology.,

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