Method for studying the interaction of a cell sample with a fluid medium

Abstract

Method for Studying the Behavior of a Cell Sample The invention relates to a method for studying the behavior of a cell sample (10) contained in a medium (25) containing a plurality of anisotropically shaped nanoparticles dispersed in this medium, the method comprising: (i) determining at least one orientation characteristic of the nanoparticles in a measurement zone at the interface between the fluid medium (25) and the cell sample (10), the orientation resulting at least partially from the interaction of the fluid medium (25) and the biological material (10), (ii) determining an average shear rate characteristic of the fluid medium in the measurement zone from said at least one nanoparticle orientation characteristic determined in this measurement zone, (iii) determining a characteristic of the cell sample (10) from the average shear rate thus determined in the measurement zone. Figure for the abstract: Fig.1.

Description

Title of the invention: Method for studying the interaction of a cell sample with a fluid medium

[0001] The present invention relates to a method for studying the behavior of a cell sample contained in a fluid medium. The invention also relates to the corresponding study device. technical field

[0002] Currently, studying the behavior of a cell sample, particularly its viability and / or functionality, most often requires the use of high-resolution optical or electron microscopy tools, combined with complex image analysis and / or genetic analyses. Such procedures are slow and expensive, making them poorly suited to clinical studies, especially in the case of in vitro evaluation of biological cells collected in routine healthcare practice.

[0003] By way of example, mucociliary clearance is one of the main defense mechanisms by which the lungs continuously clear inhaled particles from the airways. Undesirable particles are trapped by the mucus covering the airway epithelium. Airborne contaminants and mucus are then carried together along the airways by the synchronous beating of cilia present on the airway epithelium, until they are expelled into the esophagus. The absence or impairment of this clearance can lead to chronic obstructive disorders due to mucus accumulation, disorders observed not only in rare congenital diseases such as cystic fibrosis or primary ciliary dyskinesia (PCD), but also in much more common diseases such as chronic rhinitis, asthma, and chronic obstructive pulmonary disease (COPD).This absence or alteration of mucociliary clearance may be due to a dysfunction of the individual beating of the cilia (movement or motility) and / or of ciliary coordination.

[0004] In vivo observation of ciliary beating to assess mucociliary clearance is difficult. In the past, in vivo assessment of mucociliary clearance was performed using techniques involving saccharin, a drop of blue marker, or the clearance of radioactive tracers. Micro-optical coherence tomography has also been proposed. However, due to the constraints imposed by these different techniques, particularly the patient's cooperation for the saccharin test, the invasive nature of endoscopic examination, and / or the inhalation of radiopharmaceuticals, they have been virtually abandoned in practice. Current clinical practice. Currently, the most commonly used method for assessing ciliary beating is ex vivo bright-field microscopy, often combined with high-speed video microscopy (HSVM) analysis of the ciliary beating frequency of hair cells obtained by nasal or bronchial brushing. Another method used to assess mucociliary clearance involves tracking the movement of microbeads labeling the fluid displacement generated by ciliary beating using particle imaging velocimetry (PIV). This method is described in the article Bottier, M. et al. “A new index for characterizing micro-bead motion in a flow induced by ciliary beating: part II, modeling”, PLoS Comput. Biol. 13(7): el005552 (2017). This method relies on a theoretical model of the flow motion induced by the active beating of the cilia, allowing us to evaluate the shear stress exerted by the cilia on the medium.However, this shear stress proves to be a reliable indicator for characterizing the efficiency of ciliary beat. Nevertheless, this experimental method for evaluating shear has low spatial resolution and has proven to be time-consuming and difficult to implement in routine clinical practice.

[0005] Recent work described in the article Kim, J., Michelin, S., Hilbers, M. et al. “Monitoring the orientation of rare-earth-doped nanorods for flow shear tomography.” Nature Nanotech 12, 914-919 (2017) allows for mapping the shear rate in a microchannel through which a fluid flows, using europium-doped lanthanum phosphate (LaPO4:Eu) nanorods in suspension. This method has never been used to evaluate the behavior of cell samples in a fluid, and its implementation requires solving numerous problems, including the stability of the nanorods in a medium suitable for the cell samples, the non-toxicity of the nanorods to cells, and obtaining spatiotemporal resolution sufficient to study the dynamic behavior of the cell sample at the microscopic level.

[0006] There is therefore a need for a method for studying the behavior of a cell sample in a fluid, which is fast, has good resolution, is both easy to implement in clinical practice and inexpensive. Description of the invention

[0007] The invention addresses this need by a method for studying the behavior of a cell sample contained in a fluid medium containing a plurality of anisotropically shaped nanoparticles dispersed in this medium, the method comprising: (i) determining at least one orientation characteristic of the nanoparticles in a measurement zone at the interface between the fluid medium and the cell sample, the orientation resulting at least partially from the interaction of the fluid medium and the cell sample, (ii) the determination of a shear characteristic of the fluid medium in the measurement zone from the orientation characteristic of the nanoparticles determined in that measurement zone, (iii) the determination of a characteristic of the cell sample from the shear characteristic thus determined in the measurement area.

[0008] By "shear characteristic" is meant the measurement of a characteristic of the fluid velocity gradient in the fluid medium at the interface with the cell sample, in particular its direction and / or magnitude. Such a measurement makes it possible to characterize the shear force exerted by the cell sample on the fluid medium.

[0009] By "anisotropic deformed nanoparticles" is understood particles having an aspect ratio defined as the ratio of length to largest transverse dimension greater than 1, i.e. being substantially elongated in at least one direction.

[0010] By "dispersed", it is understood that the nanoparticles are suspended in the medium without aggregating together.

[0011] By "the orientation of nanoparticles," it is understood that nanoparticles, by virtue of their anisotropic shape, can orient themselves statistically within the fluid medium according to the shear stresses they experience. The higher the shear stress they experience, the more the nanoparticles will orient themselves in the same direction, corresponding approximately to the direction of fluid flow, and the narrower the orientation distribution will be around this direction.

[0012] As explained above, nanoparticles orient themselves in the fluid medium according to the shear force they experience. Determining at least one orientation characteristic of the nanoparticles in a measurement area makes it possible to determine the shear rate in the measurement area and to deduce information about a characteristic of the cell sample, in particular the overall efficiency of ciliary beating, for example by comparison with a predetermined local shear rate for a healthy cell sample.

[0013] Preferably, the process is an ex vivo or extemporaneous process.

[0014] The process is non-therapeutic as such. Cell sample

[0015] Preferably, the cell sample is composed of at least one cell cluster, in particular selected from among beating ciliated epithelial cells, especially a sample from a border of a ciliated epithelium, flagellated cells, cells embryonic cells, blood cells, reproductive cells, red blood cells, immune system cells.

[0016] The cell sample may be of human, animal, or plant origin. Fluid medium

[0017] Preferably, the fluid medium is liquid, in particular aqueous.

[0018] Preferably, the fluid medium comprises a physiologically acceptable solution For the cell sample, specifically Dulbecco's Modified Eagle Medium (DMEM), to maintain cell sample viability. If necessary, the fluid medium can contain nutrients for the cell sample and serve as a culture medium.

[0019] Preferably, the fluid circulation is induced by movement on the surface of the biological sample without external action. Such circulation can be, for example, the result of cilia movement on the surface of the cell sample. Nanoparticles

[0020] Preferably, the nanoparticles are nanorods, that is, nanoparticles of generally elongated shape extending mainly along a guideline, curvilinear or, preferably, straight. In this case, the aspect ratio is the ratio of the length, measured along this guideline, to the width, the width being the largest dimension that can be measured in all the transverse planes (perpendicular to the guideline) along the guideline.

[0021] Preferably, the aspect ratio of the nanoparticles is greater than or equal to 3, better greater than or equal to 10.

[0022] Preferably, the nanoparticles are configured to emit photoluminescent radiation and / or are birefringent.

[0023] Preferably, the nanoparticles exhibit polarized photoluminescence emission.

[0024] Preferably, the nanoparticles are rare-earth-doped nanorods. The nanoparticles may be of oxide or fluoride, in particular lanthanum phosphate (LaPO4), sodium yttrium fluoride (NaYF4), or their derivatives, doped with rare earth elements, in particular europium. The nanorods may have crystalline symmetry, in particular symmetry along an axis parallel to the nanorod's direction line. The nanorods are preferably of europium-doped LaPO4 and may be in the rhabdophan crystalline phase, or preferably in the monazite crystalline phase.

[0025] Such nanorods each emit photoluminescent radiation that is strongly polarized depending on the orientation of the nanorods. It is It is therefore possible, as described in the article by Kim, J., Michelin, S., Hilbers, M. et al., "Monitoring the orientation of rare-earth-doped nanorods for flow shear tomography," Nature Nanotech 12, 914-919 (2017), to determine at least one orientation characteristic of the nanorods by measuring the polarized fluorescence radiation of nanorods at different polarization angles within a measurement area containing several nanorods, including several hundred nanorods. A difference in the shape of the spectrum at different polarization angles is characteristic of a particular orientation of the nanoparticles and thus of the presence of a shear force in the measurement area within the fluid medium.

[0026] Such rods are also birefringent and the measurement of at least one feature of the birefringence profile in a measurement area can also make it possible to determine an orientation feature of the nano-rods and to deduce a shear feature in the measurement area.

[0027] Preferably, the nanoparticles have an average length less than or equal to 1 pm, preferably less than or equal to 200 nm. Preferably, the standard deviation of the nanoparticle length distribution is less than or equal to 100 nm. Such a nanoparticle distribution allows for good spatiotemporal measurement resolution and good sensitivity of the nanoparticle orientation to shear stress in the fluid medium.

[0028] Preferably, the nanoparticles have surface-mounted stabilizer binding molecules for dispersion stabilization, in particular binding molecules with terminal amine, silane (PEG-silane), or alendronic acid groups. Such molecules improve the dispersion stability of nanoparticles in the biological fluid medium by isolating the nanoparticle core from the fluid medium. Furthermore, they are relatively non-toxic to the cell sample.

[0029] Preferably, the concentration of nanoparticles in the fluid medium is less than or equal to 10% by volume fraction of the fluid medium. Photoluminescence

[0030] Preferably, the nanoparticles are photoluminescent and the step of determining the orientation characteristic of the nanoparticles comprises: - the photoluminescence excitation of the nanoparticles by a light source generating the emission of photoluminescence light by the nanoparticles, and - the measurement in the measurement area of ​​at least two spectral information of the photoluminescence light polarized to one or more different polarizations, - the determination of the orientation characteristic of the nanoparticles from the measurements of at least two spectral information of the photoluminescence light polarized to one or more different polarizations.

[0031] Preferably, the measurement of the two spectral data includes the optical detection of the photoluminescence light from the nanoparticles using an optical microscopy system, in particular a confocal one. The microscopy system may include a confocal optical microscope and one or more optical sensors for the light emitted by photoluminescence from the nanoparticles. The optical sensor(s) may be photodiodes, in particular photomultiplier tubes (PMTs), avalanche photodiodes (APDs), combined with spectral optical filters or a wavelength-resolved spectrometer.

[0032] The method may include the rotation of a polarizing filter to change the polarization angle, in particular arranged between the fluid medium and the optical system, in particular the optical sensor(s) to allow the measurement of at least two spectral information of the photoluminescent light polarized at one or more different polarizations.

[0033] Preferably, the excitation light source is a laser source configured to excite the nanoparticles at a predetermined optimal wavelength corresponding in particular to an excitation peak, in particular between 250 nm and 550 nm, in particular for LaPO4:Eu at a wavelength of 394 nm.

[0034] Alternatively, the excitation light source emits light having a broad wavelength spectrum, in particular white light.

[0035] In the case of LaPO4:Eu, the at least two measured spectral information can correspond to the spectra between 570 and 720 nm in which the transition bands of the Eu3+ ion are located

[0036] The method may include measuring the intensity of polarized light at one or more different polarization angles at at least two different given wavelengths, the given wavelengths corresponding in the spectrum of light emitted by the nanoparticles to two distinct intensity peaks of the photoluminescence light spectrum, in particular for LaPO4:Eu nanorods, being between 580 nm and 590 nm, and between 590 and 600 nm respectively, - between 610 nm and 617 nm and between 617 and 630 nm respectively, or - between 680 nm and 690 nm and between 690 and 710 nm respectively.

[0037] Preferably, the angle between the two polarization angles is greater than or equal to 10°, better greater than or equal to 30°, even better greater than or equal to 60°, for example equal to 90°.

[0038] The method may include identifying a difference in intensity of polarized light at two different polarization angles at a given wavelength corresponding to a preferential orientation of the nanoparticles in a particular direction.

[0039] The method may include measuring a plurality of spectral information of polarized photoluminescent light, in particular the intensity of polarized light at a given wavelength range or the spectral profile of polarized light at several different polarization angles.

[0040] Alternatively, the method comprises - the measurement of the birefringence profile of the nanoparticle distribution in a measurement area, - the determination of an orientation characteristic of nanoparticles from the measurement of birefringence of the distribution of nanoparticles in a measurement area.

[0041] Preferably, the measurement of spectral information of photoluminescent light polarized to one or more different polarizations is done temporally after the photoluminescence excitation of the nanoparticles by a light source, in particular at least 5 microseconds, better at least 10 microseconds after to eliminate parasitic fluorescence. Orientation

[0042] Preferably, the shear characteristic determined in the measurement area is the average shear value in the measurement area.

[0043] Preferably, the method comprises determining at least two characteristics of the nanoparticle orientation: the nanoparticle orientation and the associated order parameter characteristic of the dispersion of the nanoparticle orientation relative to the orientation in the measurement area. The determination method is as described in the article Kim, J., Michelin, S., Hilbers, M. et al. “Monitoring the orientation of rare-earth-doped nanorods for flow shear tomography.” Nature Nanotech 12, 914-919 (2017), incorporated herein by reference.

[0044] Preferably, the method includes determining the shear direction and shear value in the measurement area from the two orientation characteristics of the nanoparticles determined in this measurement area. Measurement zone

[0045] Preferably, the measurement area corresponds to the focal volume of the optical microscopy system. It may have a volume less than or equal to 3 pm3, preferably 1 pm3. Dynamic mode

[0046] The method may include determining the dynamic shear characteristic of the fluid medium in the measurement area by the method described above from continuous measurements over time in the same measurement area and determining a characteristic of the cell sample from the dynamic shear characteristic thus determined in the measurement area. Scan mode

[0047] The process may include: - the scanning of the fluid medium in at least two directions by the measuring system, - the determination of at least one orientation characteristic of the nanoparticles at each position of the measurement system, - the determination of a map of the shear characteristic in the fluid medium from at least one orientation characteristic of the nanoparticles measured at each position of the measurement system. Characterization of the cell sample

[0048] Preferably, the method includes comparing the shear characteristic in the determined measurement area or mapping the shear characteristic to a reference shear characteristic or a reference map of the pre-established shear characteristic, in particular for a reference cell sample.

[0049] The method may include characterization, in particular the efficiency of the cell sample from the shear characteristic in the determined measurement area or from mapping the determined shear characteristic.

[0050] Preferably, the cell sample is of ciliated epithelial cells and the method comprises determining the efficiency of the ciliary beating of said cells by comparing the determined shear rate or the determined shear rate map and a pre-established reference shear rate or map corresponding to a normal beating of the cilia of the ciliated epithelial cells.

[0051] Preferably, the cell sample is obtained by nasal brushing.

[0052] Preferably, the time between obtaining the cell sample and the measurements is less than or equal to 3 hours. Device

[0053] The invention also relates to a device for studying a cell sample, in particular for implementing the process defined above, comprising: - a fluidic chamber comprising: o a fluid medium, and o a plurality of anisotropically shaped nanoparticles dispersed in the medium, - a measurement system for at least one characteristic piece of information on the orientation of the nanoparticles in a measurement area at the interface between the fluid medium and the cell sample.

[0054] The device may include or be connected to a processor configured to determine at least one orientation characteristic of the nanoparticles in the measurement area.

[0055] Preferably, the chamber is a microfluidic chamber.

[0056] The device may include the cell sample in the fluid medium.

[0057] The properties of the process described above apply to the device, alone or in combination.

[0058] Preferably, the device includes or is connected to a processor configured to determine the shear characteristic in the measurement area from the characteristic information of the orientation of the nanoparticles determined by the measurement system in this measurement area, in particular from the orientation characteristic of the nanoparticles determined.

[0059] Preferably, the measurement system includes a scanning system in at least two directions, preferably three directions, to move the measurement system between several measurements so as to take measurements in several different measurement areas. The processor can determine a shear rate map in the fluid medium based on orientation information in order to establish a shear rate map in the fluid medium from the orientation information of the nanoparticles measured by the measurement system in different measurement areas.

[0060] Preferably, the measuring system comprises: - a photoluminescent excitation light source for nanoparticles, - an optical microscopy system, including: o a confocal optical microscope, and o one or more optical sensors for the light emitted by photoluminescence from the nanoparticles, - a rotating movable polarizing filter positioned between the chamber and the optical microscopy system, including the optical sensor(s).

[0061] The optical sensor(s) can be configured to take a measurement delayed relative to the photoluminescent excitation light source. Brief description of the drawings

[0062] [Fig-1] Fig. 1 schematically represents an example of a chamber microfluidics,

[0063] [Fig.2] [Fig.2] schematically represents an example of a device, and

[0064] [Fig.3] [Fig.3] schematically represents the beating of cilia of cells ciliated epithelial cells in three dimensions. Detailed description

[0065] An example of a method for studying a cluster of ciliated epithelial cells according to the invention is described below.

[0066] First, ciliated epithelial cells 10 are cultured in a chamber 20 containing a physiological solution 25 containing nanorods. The ciliated epithelial cells have cilia 27 on their surface which, in the case of healthy cells, exhibit a permanent, periodic movement synchronized with that of neighboring cilia and those of adjacent cells, with a phase shift that increases with distance. The movement of the cilia follows the movement of a metachronous wave, as represented in three dimensions in [Fig. 3]. The culture chamber can be a microscope slide, a Petri dish, or preferably a microfluidic chamber.

[0067] The nanorods are made of europium-doped lanthanum phosphate (LaPO4:Eu) with a rhabdophane or monazite crystalline phase, preferably monazite, and have polyethylene glycol-silane (PEG-silane) binding molecules on their surface. In physiological solution, the nanorods exhibit a length distribution characterized by an average length between 100 nm and 500 nm with a standard deviation less than or equal to 200 nm, and a cross-sectional dimension distribution characterized by an average largest dimension between 20 nm and 5 nm with a standard deviation less than or equal to 10 nm.

[0068] A measurement area of ​​the chamber 20 is then illuminated by a light source, in particular a substantially monochromatic laser 30 with a wavelength of 395 nm, the emitted light of which is conventionally transmitted to the light input of an optical microscope 35 so as to illuminate the chamber in the measurement area. The light source 30 excites the photoluminescence of the nanorods at a wavelength of 395 nm, corresponding to the excitation peak of the 7F0-5L6 transition for Eu3+. The spectra between 580 nm and 720 nm of the photoluminescence light emitted by the excited nanorods in the measurement area are then measured at two different polarization angles, here orthogonal to each other, using a measurement system 40 and a rotating mobile polarizer 50 arranged between the microfluidic chamber 20 and the measurement system 40.The light emitted by the nanorods is transmitted to the measuring system 40 via the optical microscope 35, the polarizer 50 being arranged between the optical microscope 35 and the measuring system 40. The optical microscope may include a scanning system, in particular a piezoelectric scanning device.

[0069] The measurement area has a largest dimension less than or equal to 2 pm, the measurement system having a resolution less than or equal to 1 pm3.

[0070] Scanning the microfluidic chamber in two directions using the scanning system allows measurement at each point of the measuring chamber.

[0071] The photoluminescence spectra emitted by the nanorods in each measurement zone between 580 and 720 nm at the two polarization angles then allow the determination of a parameter of order f, between 0 and 1, quantifying the local dispersion of the orientation of the nanorods, 0 corresponding to a disordered orientation of the nanorods and 1 to totally aligned nanorods, and a director H corresponding to the average orientation of the nanorods as described on pages 6 and 7 of the appendix to the article Kim, J., Michelin, S., Hilbers, M. et al. Supplementary information to “Monitoring the orientation of rare-earth-doped nanorods for flow shear tomography”. Nature Nanotech 12, 914-919 (2017).

[0072] The shear rate value in the measurement area at each point of the measurement chamber is then determined from the order parameter f and the shear orientation is given by the average orientation of the nanorods / ], as described on pages 7 to 11 of the appendix to the article Kim, J., Michelin, S., Hilbers, M. et al. Supplementary information to “Monitoring the orientation of rare-earth-doped nanorods for flow shear tomography”. Nature Nanotech 12, 914-919 (2017).

[0073] A shear map at the interface between the fluid medium and the ciliated cell(s) can then be established. Such a map is compared to the map of healthy ciliated cells, and it is then possible to determine whether the studied ciliated cells function similarly to or very differently from the healthy reference cells. Indeed, under physiological conditions, the cilia located at the apical part of the ciliated epithelial cells beat synchronously and generate a shear force on the fluid, as described in the article Bottier, M. et al. “A new index for characterizing micro-bead motion in a flow induced by ciliary beating: part II, modeling”, PLoS Comput. Biol. 13(7): M005552 (2017). Thus, when the cilia beat asynchronously or when beating is absent, the shear detected by the method described above is weak or even non-existent.The information determined by the method described therefore makes it possible to characterize the efficiency of the beating of the hair cells studied.

[0074] Alternatively, the photoluminescence spectra emitted by the nanorods in a measurement area between 580 and 720 nm at the two polarization angles are measured continuously over time with acquisitions spaced approximately Ips apart. The shear rate value and shear orientation are then determined for each measurement as described above to deduce the dynamic behavior of the cell sample 10, in particular the movement of its cilia 27, in the measurement area.

[0075] The invention is not limited to the example just described. For example, the biological material is not limited to the epithelial ciliated cells of the airways but can also be applied to the measurement of ciliated cells of the endosalpinx of the Fallopian tubes, to flagellated cells, such as sperm or embryos, or even to organisms such as paramecia (unicellular ciliated protozoan) used as a model for the study of cilia.

[0076] Alternatively, it is also possible to determine a local orientation characteristic of the nano-rods from the measurement of the birefringence of the nano-rods in the fluid medium at the interface with the biological matter, in order to deduce the local shear rate.

[0077] Alternatively, the nanoparticles are anisotropic nanoparticles other than the aforementioned nano-rod nanoparticles, the important thing being that a physical signature of the orientation of the latter exists and is sufficiently strong and characteristic to allow the identification of the general orientation of the nanoparticles and to deduce the shear rate.

[0078] Alternatively, the nanoparticles have on their surface other binding molecules allowing both good dispersion of the nanoparticles in the fluid medium and having low toxicity to biological matter or are devoid of binding molecules on their surface, in particular when the nanoparticles are in sufficiently stable suspension.

Claims

Demands

1. A method for studying the behavior of a biological material (10) contained in a medium (25) containing a plurality of anisotropically shaped nanoparticles dispersed in this medium, the method comprising: (i) determining at least one orientation characteristic of the nanoparticles in a measurement zone at the interface between the fluid medium (25) and the biological material (10), the orientation resulting at least partially from the interaction of the fluid medium (25) and the biological material (10), (ii) determining an average shear characteristic of the fluid medium in the measurement zone from said at least one nanoparticle orientation characteristic determined in this measurement zone, (iii) determining a characteristic of the cell sample (10) from the average shear rate thus determined in the measurement zone.

2. A method according to claim 1, wherein the cell sample is composed of at least one cell cluster, in particular selected from among beating ciliated epithelial cells, in particular a sample from a border of a ciliated epithelium, flagellated cells, embryonic cells, blood cells, reproductive cells, red blood cells, immune system cells.

3. A method according to any one of the preceding claims, wherein the nanoparticles exhibit polarized photoluminescence emission.

4. A method according to any one of the preceding claims, wherein the nanoparticles are rare earth-doped nanorods.

5. A process according to any one of the preceding claims, wherein the nanoparticles are of oxide or fluoride, in particular of lanthanum phosphate (LaPO4), sodium and yttrium fluorides (NaYF4) or their derivatives, doped with rare earths, in particular europium, the nanorods being in particular of europium-doped LaPO4 in rhabdophane crystalline phase, or preferably in monazite crystalline phase.

6. A method according to any one of the preceding claims, wherein the nanoparticles have an average length less than or equal to 1 pm, preferably less than or equal to 200 nm, and a standard deviation of the length distribution of the nanoparticles less than or equal to 100 nm.

7. A method according to any one of the preceding claims, wherein the aspect ratio of the nanoparticles has an aspect ratio greater than or equal to 3, preferably greater than or equal to 10.

8. A method according to any one of the preceding claims, wherein the concentration of nanoparticles in the fluid medium (25) is less than or equal to 10% by volume fraction of the fluid medium (25).

9. A method according to any one of the preceding claims, comprising determining at least two characteristics of the orientation of the nanoparticles, the orientation of the nanoparticles and the associated order parameter characteristic of the dispersion of the orientation of the nanoparticles with respect to the orientation in the measurement area.

10. A method according to claim 9, comprising determining the shear direction and shear value in the measurement zone from the two orientation characteristics of the nanoparticles determined in that measurement zone.

11. A method according to any one of the preceding claims, wherein the nanoparticles are photoluminescent and the step of determining the orientation characteristic of the nanoparticles comprises: - the photoluminescence excitation of the nanoparticles by a light source (30) generating the emission of photoluminescent light by the nanoparticles, and - the measurement in the measurement area of ​​at least two spectral information of the photoluminescent light polarized to one or more different polarizations, - the determination of the orientation characteristic of the nanoparticles from the measurements of the at least two spectral information of the photoluminescent light polarized to one or more different polarizations.

12. A method according to claim 11, wherein the nanoparticles are europium-doped LaPO4 nanorods, at least two measured spectral information corresponding to spectra between 570 and 720 nm in which the transition bands of the Eu3+ ion are located.

13. A method according to claim 11 or 12, comprising measuring the intensity of polarized light at one or more different polarization angles at at least two different given wavelengths, the given wavelengths corresponding in the spectrum of the light emitted by the nanoparticles to two intensity peaks distinct from the spectrum of photoluminescent light, in particular for europium-doped LaPO4 nanorods being between 580 nm and 590 nm, and between 590 and 600 nm respectively, between 610 nm and 617 nm and between 617 and 630 nm respectively, or between 680 nm and 690 nm and between 690 and 710 nm respectively.

14. A method according to any one of the preceding claims, comprising: - scanning the fluid medium (25) in at least two directions, preferably three directions, by the measurement system, - determining at least one orientation characteristic of the nanoparticles at each position of the measurement system, - determining a map of the shear rate in the fluid medium (25) from said at least one orientation characteristic of the nanoparticles measured at each position of the measurement system.

15. A method according to any one of the preceding claims, comprising comparing the shear characteristic in the determined measurement area or mapping the shear characteristic to a reference shear characteristic or a reference map of the pre-established shear characteristic, in particular for a reference cell sample.

16. Device for studying a cell sample, in particular for implementing the process defined above, comprising: - a fluidic chamber (20) comprising: o a fluid medium (25), o a cell sample (10) in the fluid medium (25), and o a plurality of nanoparticles dispersed in the fluid medium (25), - a measurement system for at least one characteristic piece of information on the orientation of nanoparticles in a measurement area at the interface between the fluid medium (25) and the cell sample (10).

17. Device according to claim 16, comprising or being connected to a processor configured to determine the shear characteristic in the measurement area from the characteristic information of the orientation of the nanoparticles determined by the measurement system in this measurement area, in particular from the orientation characteristic of the nanoparticles determined.

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